BACKGROUND
nonribosomal peptide synthetases , polyketide synthases  and fatty acid synthases  employ a common biosynthetic strategy to synthesize their metabolic products by stepwise condensation of simple amino or carboxylic acid monomers.
the core catalytic domains involved in the biosynthesis of the polyketide/nonribosomal peptide/fatty acid backbone moieties are ketosynthase , acyltransferase , dehydratase , enoylreductase , ketoreductase , acyl carrier protein , condensation , adenylation  and thiolation   <cit> .
apart from these core catalytic domains, a number of auxiliary functional domains, often called tailoring domains, introduce a variety of different chemical modifications to the backbone moieties of these secondary metabolites to further increase their structural diversity.
bioinformatics analysis of various catalytic domains present in nrps and pks proteins has been an area of active research in recent years  <cit> .
these studies  <cit>  have not only led to development of novel computational methods for in silico identification of secondary metabolites by genome mining  <cit> , they have also guided rational reprogramming of secondary metabolite biosynthetic pathways to generate designed "natural products"  <cit> .
however, all these studies including our earlier work have concentrated on core catalytic domains and no detailed bioinformatics analyses have been carried out for important tailoring enzymes like, methyltransferases.
methyltransferase  domains present in nrps and pks clusters constitute a major class of tailoring domains/enzymes involved in biosynthesis of secondary metabolites.
they catalyze the transfer of methyl group from s-adenosylmethionine  to the carbon, nitrogen or oxygen atoms at various positions on the backbones of polyketides, nonribosomal peptides and fatty acids and therefore have been classified as c-mt, n-mt and o-mt respectively depending upon their site of methylation.
these enzymatic domains in general have a bidomain structure, where the first subdomain contains the binding site for methyl group donor, while the second subdomain harbors the binding site for acceptor substrate  <cit> .
the presence of mt domains in multifunctional nrps and pks proteins is generally inferred from chemical structure of the secondary metabolite products.
there are only few in vitro studies on enzymatic characterization of nrps/pks mt domains  <cit> .
a recent study on mt domains from type ii pks biosynthetic pathways has revealed interesting correlation between regioselectivity of methylation and mt sequence  <cit> .
however, no such analysis has been carried out for mt domains present in type i pks or nrps proteins.
in contrast to type ii pks mts which are stand alone proteins, mt domains in type i pks and nrps are present along with other catalytic domains on a single polypeptide chain.
therefore, it has been difficult to decipher the correct length and domain boundaries for mt domains in type i pks or nrps proteins.
various studies have suggested that the size of n-mt domain is typically  <dig> amino acids, while c-mt and o-mt are generally  <dig> amino acids long.
a set of  <dig> conserved sequence motifs has been identified in most mts  <cit> .
mutational studies of n-mts of peptide synthetases have shown that these  <dig> motifs are essential for the catalysis  <cit> .
the knowledge of these mt sequence motifs and the expected spacing between them is often used for discerning presence of mt domains in multifunctional nrps and pks proteins.
however, because of the high degree of sequence divergence, delineating the correct boundary of these proteins is quite often a difficult task.
in our earlier study, we attempted to identify mt domains in various nrps/pks gene clusters based on pairwise alignment with mt domain from actinomycin cluster  <cit> .
however, this domain identification protocol failed to detect  <dig> out of  <dig> mt domains.
the  <dig> unidentified mt domains included the three groups of mts , for which proper templates were not available.
the general purpose domain identification tools like cdd-search can identify mt domains in nrps and pks proteins, but can not predict the domain boundaries accurately and they also fail to classify them as c-mt, n-mt and o-mt.
such classification is crucial for prediction of chemical structures of secondary metabolites.
the knowledge of substrate specificity and domain boundaries of mt domains is also important for rational design of novel secondary metabolites by introduction of heterologous mt domains.
in this manuscript, we have carried out a systematic analysis of the sequence/structural features of mt domains present in various experimentally characterized nrps and type i pks clusters having known metabolic products.
since crystal structures are available for many stand alone small molecule methyltransferases from several microbial organisms, we have carried out threading analysis for the experimentally characterized mt domains from nrps and pks biosynthetic pathways.
the threading analysis has helped in elucidating the putative three dimensional structure adopted by mt domains and based on the alignment of mt containing sequences on the structural fold of mt domain it has been possible to delineate the correct boundaries for nrps/pks mt domains.
our threading analysis has also given novel insight into the structural features of linker sequences flanking the mt domains in nrps and pks proteins.
using the curated sequences of these mts, we have carried out detailed phylogenetic analysis to investigate whether these catalytic domains cluster as per their specificity for site of methylation i.e.
c-mt, n-mt and o-mt.
based on this analysis, we have identified suitable template sequences of c-mt, n-mt and o-mt domains from representative clusters, which can be used to identify mt domains in uncharacterized nrps/pks proteins.
we have also developed hidden markov model  profiles which can identify mt domains in a query sequence and classify them as n-mt, c-mt and o-mt.
using these hmm profiles, we have analyzed non-redundant protein sequence database of ncbi to identify other multifunctional enzymes containing c-mt, n-mt and o-mt domains.
RESULTS
in this study, we have carried out a number of different bioinformatics analyses on mt domains present in type i pks and nrps proteins, to correlate the sequence of these mt domains to their substrate specificity i.e.
the site of methylation.
figure  <dig> gives a schematic overview of the various different analyses carried out and the type of results obtained from them, while the results are discussed in detail in the following sections.
the chemical structures of the secondary metabolites produced by various pks, nrps and hybrid nrps/pks clusters cataloged in nrps-pks web resource were analyzed carefully to identify methyl substitutions on polyketide or nonribosomal peptide backbones.
the presence of methyl substitutions on nitrogen and oxygen atoms indicated presence of n-mt or o-mt domains in the proteins encoded by these gene clusters.
however, in absence of mt domains methyl substitutions on carbons in a ketide group can also result from selection of methylmalonate extender groups by the at domains of pks proteins.
therefore, for correctly inferring presence of c-mt domains in a pks protein, the substrate specificity of the corresponding at domain was also checked.
table  <dig> lists various orfs harboring the mt domains, their genbank accession number and the type or substrate specificity of mt domain as deduced from the chemical structure of the metabolite.
as can be seen, the data set consisted of  <dig> c-mt,  <dig> o-mt and  <dig> n-mt domains from  <dig> different nrps/pks clusters.
figure  <dig> shows the chemical structures of  <dig> representative secondary metabolites highlighting the methyl groups added by c-mt, n-mt and o-mt domains, while chemical structures of the remaining  <dig> secondary metabolites are shown in additional file  <dig>  each of the orfs listed in table  <dig> were analyzed by nrps-pks search tool as well as cdd server of ncbi.
nrps-pks search tool, which used a single mt domain from actinomycin cluster as template, could identify only  <dig> mt domains out of a total of  <dig>  even though the latest version of cdd server could identify  <dig> out of these  <dig> mt domains, the lengths of the mt domains detected by both these programs were notably shorter than the typical length of these domains.
these programs also failed to distinguish between c-mt, n-mt and o-mt domains.
additional file  <dig> shows the length of each mt containing sequence stretch and other catalytic domains flanking this region.
as can be seen, all n-mt domains are present in nrps clusters only as c-a-mt-t modules and typically a  <dig> amino acid sequence stretch containing this domain is inserted in the adenylation domain between the conserved motifs a <dig> and a <dig> of adenylation domains and hence alignment of these n-mt containing a domains with regular a domains produces a split alignment .
all stand alone o-mt containing sequences were typically  <dig> to  <dig> amino acids long, while the amino acid stretches containing other o-mt and c-mt domains present in type i pks proteins were  <dig> to  <dig> amino acids long.
the o-mt and c-mt domains in pks proteins were present in four different types of modules i.e.
ks-at-mt-acp, ks-at-mt-kr-acp, ks-at-dh-mt-kr-acp and ks-at-dh-mt-er-kr-acp.
only in case of leinamycin gene cluster, which has trans-at domains, the mt domain is present as ks-dh-kr-acp-mt-acp.
there were only two examples where mt domain was adjacent to an aminotransferase  domain in a hybrid nrps/pks system.
in these cases mt-amt stretch was inserted between a pks and a nrps module.
it appears that mt domains in type i pks proteins are present in at-kr, dh-er or dh-kr linker regions which are typically more than  <dig> amino acids long.
hence, length of the flanking linker region could be the reason for the larger length of these mt containing sequence stretches.
therefore, we decided to carry out various structure based sequences analysis for representative mt containing stretches of each category to delineate the exact domain boundaries for mt domains.
aaf42473
cac01604
cac01607
aak81826
caa82227
caa79245
t30289
aad55801
aag27087
aag27088
bac20564
bac20566
caa42929
aav66106
aad43562
q9y8a5
aad34559
cad19088
cad19089
cad19094
aag02357
aan32980
aan32981
aaf26922
aas98781
aas98785
aan85523
cad89776
cad89777
bab12210
bab12211
bab12213
baa83992
aaf19813
aaf19814
aao64404
aao64405
aao64407
aao64403
aao64406
aav97870
aav97872
aav97875
aav97876
aav97877
aas47564
aas47557
aas47560
caf05651
caf05647
caf05648
aac69588
list of orfs containing methyltransferase domains in various experimentally characterized nrps/pks gene clusters.
table also lists the number and type of mt domains in each of the orfs and results from pfam analysis using cdd search.
threading analysis of mt domains
since domain boundaries can be identified correctly by aligning the sequence of multi domain proteins with the 3d structures of the corresponding single domain proteins, we attempted to identify other proteins in pdb which are structurally similar to these mt domains of pks/nrps enzymes.
however, the lack of crystal structures for any mt domains from pks/nrps biosynthetic pathway and high degree of sequence divergence in this enzyme family prompted us to use threading or fold recognition approach.
these tools can potentially reveal structural similarity in absence of high degree of similarity in sequences.
genthreader and phyre fold prediction servers were used for threading analysis.
as discussed in the methods section, the mt sequence stretch identified by cdd or nrps-pks along with their flanking linkers was threaded on various structural folds in pdb.
in cases where chemical structure of metabolite indicated presence of mt domains but no mt domain was detected by these programs, all linker stretches having unusual length were analyzed by both these fold recognition servers.
column  <dig> gives the unique name assigned to each mt containing sequence stretch, while the second column indicates their length.
column 3– <dig> lists the pdb ids for the structures which show alignment with these sequences in fold recognition analysis using genthreader and phyre servers.
results from genthreader corresponding to confidence level certain and high are labeled as c and h respectively.
similarly, phyre results corresponding to precision level 100% and 95% are labeled as  <dig> and  <dig> respectively in curly braces.
indicates the absence of that particular fold.
it may be noted that the small molecule mt structures present in pdb show significant variation in their length and in case of some of these sequences alignments were found with mt structures having significant difference in length.
for example,  <dig> of the c-mt domains and  <dig> n-mt domain in our data set aligned with aclacinomycin-10-hydroxylase   <cit>  and histamine n-mt .
the huge length difference between these two mt structures made the choice of correct structural template a difficult task.
1qzz aligns with the beginning of the mt containing sequence stretch, while in the alignment of the same sequences with 1vlm, the length of the linker preceding the mt domain ranges from  <dig> to even  <dig> residues .
however, careful manual analysis of the alignments as well as the corresponding structural templates indicated that, the crystal structure 1qzz consists of a predominantly α-helical n-terminal domain which is involved in dimerization and a c-terminal sam binding domain typical of methyltransferases.
on the other hand, the crystal structure 1vlm contains the sam dependent mt domain alone.
the overlapping region of both the alignments correspond to the catalytic domain of methyltransferase, while the linker sequences preceding the mt catalytic domain showed alignment with additional dimerization domains, if present in the structural template.
earlier studies on structural analysis of small molecule methyltransferases have suggested that, these enzymes have a conserved sam-mt fold consisting of alternating β strands and α helices  <cit> .
earlier bioinformatics analysis  <cit>  of mt domains has also revealed that, despite high divergence in primary sequence, certain conserved sequence motifs are present in all sam dependent methyltransferases.
therefore, we wanted to examine whether the conserved secondary structural elements and sequence motifs responsible for catalytic activity, substrate specificity and cofactor binding are conserved in the mt domains identified by threading alignments.
figures  <dig>   <dig> and  <dig> show the structure guided multiple alignment of representative n-mt, c-mt and o-mt sequences with the structural template 1vlm.
as can be seen from figure  <dig>  the n-mt sequences have the conserved motifs i, ii/y, iv and v in n-mts.
similarly motifs i, motif i-post, ii and iii are present in the c-mt and o-mt sequences .
thus all the motifs  <cit>  identified in earlier analysis of small molecule methyltransferase sequences were found to be conserved in the multiple sequence alignments of c-, o- and n-mt domains identified by our threading analysis.
the average percent identity among the c-, o- and n-mt domains was found to be  <dig>   <dig> and 17% respectively.
the threading analysis also showed statistically significant matches with proteins other than methyltransferases.
such matches were specifically seen for mt containing sequences which were longer in length due to the presence of large flanking linker sequences.
as can be seen from table  <dig> and figure  <dig>  the n-terminal region of mt containing sequence from bleomycin showed an alignment  with the last  <dig> amino acids of the ks-at di-domain structure from erythromycin pks  <cit> .
this stretch corresponds to the last helix of the at domain and a segment of the at-dh linker region .
the c-terminal  <dig> amino acid stretch of the mt containing sequence from bleomycin  <cit> , nodularin and melithiazol also showed highly significant alignments  with the structural subdomain  of the recently elucidated structure of kr from erythromycin pks.
these results suggest that, in type i pks proteins, the linker sequences preceding the mt domain i.e.
at-mt linkers are homologous to the at-dh linkers and mt-kr linker regions are likely to adopt a short chain reductase  fold and would constitute the structural half of the kr domain as demonstrated in erythromycin pks.
thus mt catalytic domain is likely to be  <dig> amino acids only.
development of a computational protocol for identifying mt domains and their classification as c-mt, o-mt and n-mt
the domain boundaries were thus identified with 1vlm  and 1vl <dig>  as templates and the regions which aligned with these templates were extracted from the  <dig> mt domain sequences.
they represented the curated mt domains with correct boundary.
a set of  <dig> mt domains out of these  <dig> sequences were used as test set to check if mt domains can be correctly classified by pairwise alignment with these  <dig> template sequences.
each of the  <dig> mt domains was queried against  <dig> mt templates to find the number of mt domains identified by these templates.
the query mt domain was classified as c-mt, o-mt or n-mt depending on the highest scoring match from the template set.
the results obtained by the pairwise comparison of  <dig> query sequences with the  <dig> template sequences indicated that c-mt templates were able to identify all other c-mt sequences.
however, there were a few o- and n-mts, which were also recognized by these c-mt templates.
specifically,  <dig> mt sequences in onnamide gene cluster  showed very high similarity to c-mt while they are functionally annotated as o-mts by the authors who reported experimental characterization of this gene cluster  <cit> .
these  <dig> o-mts also have motif i as exgxg which is characteristic of c-mts.
an n-mt from pyochelin synthetase  also exhibited considerable similarity to several c-mts and the two onnamide o-mts.
in view of this apparent anomaly in sequence similarity of these proteins, their functional assignment needs to be examined carefully.
in the orf apdb of anabaenopeptilide gene cluster, there are two mt-domains wherein the first one is entirely different from all the other mt sequences and does not show similarity with any other mt sequence.
a dendrogram  of all the  <dig> mt sequences also illustrates the same pattern of results as obtained by these pairwise alignments.
the two onnamide o-mts in onnb and onni genes and the n-mts in pyochelin and anabaenopeptilide show clustering with c-methyltransferases.
a stand alone o-mt in stigmatellin  is sequentially different from other o-methyltransferases, which was observed from the pairwise alignment results and is evident from the dendrogram.
thus it can be concluded from the above analysis that these  <dig> sequences can be used as templates to identify mt-domains in any given query sequence by pairwise alignment.
these new mt templates were included in the current version of nrps-pks program for correct identification of various types of mt domains.
profile hmms of c-, o- and n-methyltransferases
an alternative approach to detect mt domains in a new sequence is to query that sequence against a database of hmm profiles.
individual profiles were built for c-mt, o-mt and n-mts using the curated sequences.
these profiles were then used to make a hmm profile database for mt domains.
the set of  <dig> c-mt, o-mt and n-mt sequences from experimentally characterized nrps/pks clusters were queried against this hmm database and the location of the mt-domain and their class was predicted in these sequences.
table  <dig> lists the score and e-value for alignment of  <dig> representative mt sequences with the hmm profiles of n-mt, c-mt and o-mt domains.
as can be seen from table  <dig>  most c-mt sequences show statistically significant alignment with o-mt profiles and vice versa.
on the other hand, only the n-mt sequences from pyochelin aligned with the n-mt as well as c-mt profiles unlike the other n-mt sequences which aligned with n-mt profile alone.
this finding is consistent with results from pairwise sequence alignment discussed in the previous section.
a '-'sign indicates that alignments resulted in scores with e-value higher than  <dig> e- <dig>  several c-mts align with o-mt profiles, while only n-mt of pyochelin synthase shows alignment with c-mt profile as well.
in order to test the predictive ability of our mt hmm profiles further, the recent version of the nr database of ncbi was also searched using these profiles to identify putative nrps/pks mt domains in various proteins.
the sequences which matched with these profiles were grouped as c-mt, o-mt and n-mt containing sequences based on highest scoring profile match.
the complete domain architecture of these mt containing proteins were also analyzed in details.
the nr database search identified  <dig> stand alone mt proteins and  <dig> multifunctional proteins containing mt domains.
out of the  <dig> stand alone mt proteins,  <dig> were o-mt proteins.
in contrast to the very large number of stand alone o-mt proteins, there were only  <dig> stand alone c-mt and  <dig> stand alone n-mt proteins.
even though experimentally characterized nrps/pks biosynthetic pathways have a relatively larger number of stand alone o-mt proteins compared to stand alone c-mt or n-mt domains, it is not apparent whether all these stand alone o-mt proteins identified by our profile search would indeed be associated with secondary metabolite biosynthesis.
analysis of domain architectures in multifunctional proteins containing mt domains revealed several interesting results.
these mt containing multifunctional proteins can be divided into four major groups.
they are proteins containing mt domains along with core pks domains, core nrps domains, pks as well nrps domains and other catalytic domains.
figure  <dig> shows the number of proteins containing n-mt, o-mt and c-mt domains for each of the four categories.
as can be seen from figure 10a,  <dig> pks proteins have c-mt domains,  <dig> pks proteins have o-mt and none of them have n-mt domains.
this result is consistent with the fact that polyketides have no sites for n-methylation and o-methylation in known pks biosynthetic pathways is catalyzed by stand alone o-mts.
this indicates that our profiles are able to correctly classify the three different classes of secondary metabolite methyltransferases.
our nr database search also identified  <dig> nrps proteins containing n-mt domains,  <dig> containing o-mt domains and  <dig> containing c-mt domains .
since most nonribosomal peptide products are n-methylated, the presence of such a large number of c-mt domains as c-a-cmt-t modules was surprising.
similarly, the hybrid nrps/pks set also had  <dig> proteins containing c-mt domains as compared to only  <dig> proteins containing n-mt domains .
in fact  <dig> of these c-mt domains in hybrid nrps/pks proteins were present next to condensation domains of nrps as c-cmt-pcp modules, while one would a priori expect n-mt domains in such modules instead of c-mt domains.
in view of this finding of anomalously large number of nrps modules containing c-mt domains in nrps and hybrid nrps/pks proteins, we decided to analyze these proteins by pairwise alignment with  <dig> representative mt templates from known nrps/pks biosynthetic pathways.
interestingly the n-mt domain of pyochelin synthase was found to be closest match for  <dig> out of the  <dig> c-mt containing nrps proteins identified by profile search.
most of these  <dig> nrps proteins showed very high percentage identity with pyochelin synthase n-mt ranging from 27% to 69%.
thus it is very much likely that, these  <dig> nrps proteins indeed contain pyochelin type n-mt domains which are different from other n-mt domains.
they were annotated by profile approach as c-mts, because n-mt domain of pyochelin synthase shows homology to c-mts and has comparable scores with c-mt as well as n-mt profiles .
similarly, a c-mt domain from yersiniabactin synthase was found to be the closest homolog of c-mts found in nrps modules of hybrid nrps/pks proteins and the sequence similarity was also very high.
this mt domain in yersiniabactin synthase catalyzes c-methylation , but is present as c-cmt-pcp module similar to the domain organization found in c-mt containing hybrid nrps/pks proteins found by our profile search.
this suggests that our profile search has genuinely identified yersiniabactin type novel c-mt domains embedded in nrps modules.
in case of  <dig> other nrps proteins which showed matches with c-mt profiles, the c-mt domains from yersiniabactin, leinamycin and nodularin were found to be closest match.
table  <dig> shows the domain organization predicted for each of them by hmm profiles, and the percentage identity and similarity with closest matching mt domain in our  <dig> representative templates set.
even though the closest homolog approach also detects c-mt domains in these proteins in agreement with results from hmm approach, in view of the relatively low sequence identity with c-mts from known nrps/pks biosynthetic pathways, it is not clear if these domains are likely to be genuine c-mt domains as in yersiniabactin synthase or a different class of n-mt domains which lack homology to known n-mt domains in our data set.
thus our analysis of nr database has revealed presence of many c-mt domains in nrps modules as in yersiniabactin.
it has also identified several pyochelin type n-mt domains which often show higher homology to c-mt profiles.
none of these proteins are currently experimentally characterized.
experimental characterization of some of these mt domains would help in understanding how mt domains in nrps/pks family have evolved to acquire specificities for different substrates.
however, a close examination of the chemical structures of pyochelin  <cit>  and yersiniabactin  <cit>  provides a rational for presence of n-mt domains with homology to c-mt sequences.
as can be seen from figure  <dig>  in both these biosynthetic clusters mt domains transfer methyl groups to a five membered rings having very similar chemical structures.
they only differ by the site of methylation.
in view of the similarity in the structure of the acceptor substrate, pyochelin type n-mt domains show homology to c-mt proteins.
it may be interesting to note that, in a recent study involving mts from type ii pks pathways, hertweck and coworkers  <cit>  have observed that, these type ii mts cluster according to the site of methylation not necessarily as c-mts, o-mts and n-mts.
based on this observation, they have proposed that for polyketide alkylation, regioselectivity is more dominant than the type of nucleophile  that is being alkylated.
thus the c-mt domains found in nrps modules by our profile search could indeed be due to such correlation between sequence of type i mts and their regioselectivity.
these observations have interesting implications of prediction of chemical structures of metabolites by genome mining.
list of protein sequences from nr database which contain c-mt domains adjacent to the core nrps domains as identified by our mt hmm profile search.
gene identifier number, domain organization and organism name is listed in columns  <dig>   <dig> and  <dig>  columns  <dig> and  <dig> list the closest match  with the representative mt domains from experimentally characterized nrps/pks clusters and the corresponding percentage identities.
the analysis of mt containing proteins in nr database also revealed the co-occurrence of nrps/pks type mt domains with several other catalytic domains apart from core pks and nrps domains .
however, in most cases these proteins had o-mt domains and there were relatively few c-mt and n-mt domains.
table  <dig> shows domain organization for representative proteins from each category and also lists the number of such proteins identified by our hmm profile search.
as can be seen, the o-mt domains are present along with a variety of catalytic domains which do not belong to core nrps or pks family.
interestingly  <dig> out of the  <dig> examples correspond to co-occurrence with glycosyltransferase domains.
since glycosyltransferases are a major class of tailoring enzymes involved in secondary metabolite biosynthesis, many of these proteins might indeed be associated with novel pks/nrps biosynthetic pathways  <cit> .
apart from glycosyltransferases, the other functional domains which occur with o-mt are oxidases, hydroxylases, tetratricopeptide repeat , phosphodiasterases, dna binding helix-turn helix proteins etc.
it would be necessary to further analyze each of these proteins in details to understand their biochemical function.
in contrast to o-mts, the n-mt and c-mt domains are present in multifunctional enzymes which contain other catalytic domains in addition to pks and nrps domains.
some of the interesting examples are nrps/pks proteins containing chalcone synthase or carnitine acyltransferase domains along with c-mt domains.
similarly, n-mt domains are present along with domains associated with streptococcal surface antigen, bacterial luciferase, phosphoenol pyruvate  utilizing enzyme and ketopantoate reductase  etc.
thus these results give valuable clues about organisms which can potentially make secondary metabolites with a variety of structural modifications by diverse types of tailoring enzymes.
some of these proteins would be interesting targets for detailed experimental investigation.
list of representative protein sequences from nr database which contain c-mt, n-mt or o-mt domains in combination with functional domains other than core pks or nrps domains.
gene identifier number, domain organization and organism names are listed in columns  <dig>   <dig> and  <dig> respectively, while column  <dig> lists total number of proteins having similar domain organization.
discussions
we have carried out a comprehensive analysis of the sequence and structural features of the mt domains present in multi functional proteins encoded by various experimentally characterized nrps, pks and hybrid nrps/pks gene clusters with known secondary metabolite products.
even though presence of methyltransferase domains in nrps and pks family of megasynthases have been inferred from methylation pattern of the chemical structure of the secondary metabolite product, earlier studies have not defined the correct domain boundaries.
threading analysis of the mt containing sequence stretches suggest 1vlm and 1vl <dig> as possible structural templates for nrps/pks mt domains.
based on the alignment with these structural templates, we identify the correct boundaries and predict that, the general length of mt domains will be in the range of 200– <dig> residues.
our threading analysis also reveals interesting homology between at-mt and at-dh linkers.
similarly, large sequence stretches c-terminus to the c-mt domains of pks proteins are found to have homology with structural half of kr domains.
these results not only explain the large variation in the length of the mt containing sequence stretch, they can also provide valuable clues for design of domain swapping experiments.
the curated sequences of these mt domains were further analyzed.
from the mt sequences of correct length, a representative set of  <dig> mt domains covering the entire range of sequence divergence were chosen.
a novel protocol for identification of mt domains by pairwise alignment and their classification as n-mt, c-mt and o-mt was developed using these  <dig> mts as multiple templates.
this mt domain identification protocol has been implemented in the current version of the program nrps-pks.
using this approach c-mt domains were annotated in the pks proteins of dictyostelium discoideum in a recent work  <cit> .
the mt sequences with correct domain boundaries were also used to build profile hmms for o-mt, c-mt and n-mt domains.
using these hmm profiles searches were carried out in the nr database of ncbi for identifying various other proteins containing c-mt, o-mt and n-mt domains.
it is interesting to note that, apart from core pks and nrps domains, these secondary metabolite biosynthetic mt domains are also associated with other important catalytic domains like glycosyltransferase, oxidases, hydroxylases, phosphodiesterases and reductases.
these proteins could be interesting targets for experimental characterization.
our analysis also surprisingly revealed the presence of a large number c-mt domains in nrps modules adjacent to condensation  and adenylation  domains.
these predicted c-mt domains can be classified into two groups.
one group of c-mt domains showed high homology to n-mt domain of pyochelin synthase, which is different from typical n-mt domains present in nrps modules.
unlike typical n-mt domains of nrps proteins, this shows homology to c-mt domains but catalyzes transfer of methyl group to nitrogen.
this group could indeed be pyochelin type n-mt domains, but are mis-classified as c-mt due to their homology with c-mt proteins and under representation of pyochelin type n-mt in our training data set.
the closest homolog of the other group of c-mt domains present in nrps modules is the c-mt domain of yersiniabactin synthase, which is present adjacent to a condensation domain of nrps, but is indeed a c-mt.
however, in view of the low homology between the c-mt from yersiniabactin and this second group of predicted c-mts, it is difficult to predict whether they are yersiniabactin or pyochelin type mts.
a close examination of the chemical structures of the yersiniabactin and pyochelin provides an evolutionary basis for the presence of pyochelin type n-mt domains having homology with c-mt proteins.
as can be seen from figure  <dig>  an identical five membered ring is the acceptor moiety for these two classes of mts, while pyochelin n-mt methylates at the n position of the ring, the yersiniabactin c-mt methylates the adjacent c position.
it may be interesting to note that such correlation between regioselectivity of the site of methylation and mt sequence have also been reported earlier by hertweck and coworkers  <cit>  for mts in type ii pks biosynthetic pathways.
experimental characterization of proteins identified by our analysis would help in building more specific profiles for these novel mt domains.
CONCLUSIONS
we have carried out a comprehensive bioinformatics analysis of methyltransferase  domains present in pks/nrps clusters having known secondary metabolite products.
based on the site of methylation of these known secondary metabolites, the mt domains have been grouped as n-mt, c-mt and o-mt proteins and sequence/structural features have been analyzed in detail for each group.
based on the results of this analysis, we have developed a novel knowledge based computational approach for detecting mt domains present in pks and nrps megasynthases, delineating their correct boundaries and classifying them as n-mt, c-mt and o-mt using profile hmms.
analysis of proteins in nr database of ncbi using these class specific profiles has revealed several interesting examples of mt domains with novel substrate specificities.
our analysis has also given interesting insight into the evolutionary basis of the novel substrate specificities of these mt proteins.
these results have interesting implications for identification of novel secondary metabolites by genome mining and also rational design of novel natural products by biosynthetic engineering.

 nonribosomal peptide synthetases, polyketide synthases and fatty acid synthases employ a common biosynthetic strategy to synthesize their metabolic products by stepwise condensation of simple amino or carboxylic acid monomers. the core catalytic domains involved in the biosynthesis of the polyketide/nonribosomal peptide/fatty acid backbone moieties are ketosynthase, acyltransferase, dehydratase, enoylreductase, ketoreductase, acyl carrier protein, condensation, adenylation and thiolation. apart from these core catalytic domains, a number of auxiliary functional domains, often called tailoring domains, introduce a variety of different chemical modifications to the backbone moieties of these secondary metabolites to further increase their structural diversity. bioinformatics analysis of various catalytic domains present in nrps and pks proteins has been an area of active research in recent years. these studies have not only led to development of novel computational methods for in silico identification of secondary metabolites by genome mining, they have also guided rational reprogramming of secondary metabolite biosynthetic pathways to generate designed "natural products". however, all these studies including our earlier work have concentrated on core catalytic domains and no detailed bioinformatics analyses have been carried out for important tailoring enzymes like, methyltransferases. methyltransferase domains present in nrps and pks clusters constitute a major class of tailoring domains/enzymes involved in biosynthesis of secondary metabolites. they catalyze the transfer of methyl group from s-adenosylmethionine to the carbon, nitrogen or oxygen atoms at various positions on the backbones of polyketides, nonribosomal peptides and fatty acids and therefore have been classified as c-mt, n-mt and o-mt respectively depending upon their site of methylation. these enzymatic domains in general have a bidomain structure, where the first subdomain contains the binding site for methyl group donor, while the second subdomain harbors the binding site for acceptor substrate. the presence of mt domains in multifunctional nrps and pks proteins is generally inferred from chemical structure of the secondary metabolite products. there are only few in vitro studies on enzymatic characterization of nrps/pks mt domains. a recent study on mt domains from type ii pks biosynthetic pathways has revealed interesting correlation between regioselectivity of methylation and mt sequence. however, no such analysis has been carried out for mt domains present in type i pks or nrps proteins. in contrast to type ii pks mts which are stand alone proteins, mt domains in type i pks and nrps are present along with other catalytic domains on a single polypeptide chain. therefore, it has been difficult to decipher the correct length and domain boundaries for mt domains in type i pks or nrps proteins. various studies have suggested that the size of n-mt domain is typically amino acids, while c-mt and o-mt are generally amino acids long. a set of conserved sequence motifs has been identified in most mts. mutational studies of n-mts of peptide synthetases have shown that these motifs are essential for the catalysis. the knowledge of these mt sequence motifs and the expected spacing between them is often used for discerning presence of mt domains in multifunctional nrps and pks proteins. however, because of the high degree of sequence divergence, delineating the correct boundary of these proteins is quite often a difficult task. in our earlier study, we attempted to identify mt domains in various nrps/pks gene clusters based on pairwise alignment with mt domain from actinomycin cluster. however, this domain identification protocol failed to detect out of mt domains. the unidentified mt domains included the three groups of mts, for which proper templates were not available. the general purpose domain identification tools like cdd-search can identify mt domains in nrps and pks proteins, but can not predict the domain boundaries accurately and they also fail to classify them as c-mt, n-mt and o-mt. such classification is crucial for prediction of chemical structures of secondary metabolites. the knowledge of substrate specificity and domain boundaries of mt domains is also important for rational design of novel secondary metabolites by introduction of heterologous mt domains. in this manuscript, we have carried out a systematic analysis of the sequence/structural features of mt domains present in various experimentally characterized nrps and type i pks clusters having known metabolic products. since crystal structures are available for many stand alone small molecule methyltransferases from several microbial organisms, we have carried out threading analysis for the experimentally characterized mt domains from nrps and pks biosynthetic pathways. the threading analysis has helped in elucidating the putative three dimensional structure adopted by mt domains and based on the alignment of mt containing sequences on the structural fold of mt domain it has been possible to delineate the correct boundaries for nrps/pks mt domains. our threading analysis has also given novel insight into the structural features of linker sequences flanking the mt domains in nrps and pks proteins. using the curated sequences of these mts, we have carried out detailed phylogenetic analysis to investigate whether these catalytic domains cluster as per their specificity for site of methylation i.e. c-mt, n-mt and o-mt. based on this analysis, we have identified suitable template sequences of c-mt, n-mt and o-mt domains from representative clusters, which can be used to identify mt domains in uncharacterized nrps/pks proteins. we have also developed hidden markov model profiles which can identify mt domains in a query sequence and classify them as n-mt, c-mt and o-mt. using these hmm profiles, we have analyzed non-redundant protein sequence database of ncbi to identify other multifunctional enzymes containing c-mt, n-mt and o-mt domains.  in this study, we have carried out a number of different bioinformatics analyses on mt domains present in type i pks and nrps proteins, to correlate the sequence of these mt domains to their substrate specificity i.e. the site of methylation. figure gives a schematic overview of the various different analyses carried out and the type of results obtained from them, while the results are discussed in detail in the following sections. the chemical structures of the secondary metabolites produced by various pks, nrps and hybrid nrps/pks clusters cataloged in nrps-pks web resource were analyzed carefully to identify methyl substitutions on polyketide or nonribosomal peptide backbones. the presence of methyl substitutions on nitrogen and oxygen atoms indicated presence of n-mt or o-mt domains in the proteins encoded by these gene clusters. however, in absence of mt domains methyl substitutions on carbons in a ketide group can also result from selection of methylmalonate extender groups by the at domains of pks proteins. therefore, for correctly inferring presence of c-mt domains in a pks protein, the substrate specificity of the corresponding at domain was also checked. table lists various orfs harboring the mt domains, their genbank accession number and the type or substrate specificity of mt domain as deduced from the chemical structure of the metabolite. as can be seen, the data set consisted of c-mt, o-mt and n-mt domains from different nrps/pks clusters. figure shows the chemical structures of representative secondary metabolites highlighting the methyl groups added by c-mt, n-mt and o-mt domains, while chemical structures of the remaining secondary metabolites are shown in additional file each of the orfs listed in table were analyzed by nrps-pks search tool as well as cdd server of ncbi. nrps-pks search tool, which used a single mt domain from actinomycin cluster as template, could identify only mt domains out of a total of even though the latest version of cdd server could identify out of these mt domains, the lengths of the mt domains detected by both these programs were notably shorter than the typical length of these domains. these programs also failed to distinguish between c-mt, n-mt and o-mt domains. additional file shows the length of each mt containing sequence stretch and other catalytic domains flanking this region. as can be seen, all n-mt domains are present in nrps clusters only as c-a-mt-t modules and typically a amino acid sequence stretch containing this domain is inserted in the adenylation domain between the conserved motifs a and a of adenylation domains and hence alignment of these n-mt containing a domains with regular a domains produces a split alignment. all stand alone o-mt containing sequences were typically to amino acids long, while the amino acid stretches containing other o-mt and c-mt domains present in type i pks proteins were to amino acids long. the o-mt and c-mt domains in pks proteins were present in four different types of modules i.e. ks-at-mt-acp, ks-at-mt-kr-acp, ks-at-dh-mt-kr-acp and ks-at-dh-mt-er-kr-acp. only in case of leinamycin gene cluster, which has trans-at domains, the mt domain is present as ks-dh-kr-acp-mt-acp. there were only two examples where mt domain was adjacent to an aminotransferase domain in a hybrid nrps/pks system. in these cases mt-amt stretch was inserted between a pks and a nrps module. it appears that mt domains in type i pks proteins are present in at-kr, dh-er or dh-kr linker regions which are typically more than amino acids long. hence, length of the flanking linker region could be the reason for the larger length of these mt containing sequence stretches. therefore, we decided to carry out various structure based sequences analysis for representative mt containing stretches of each category to delineate the exact domain boundaries for mt domains. aaf42473 cac01604 cac01607 aak81826 caa82227 caa79245 t30289 aad55801 aag27087 aag27088 bac20564 bac20566 caa42929 aav66106 aad43562 q9y8a5 aad34559 cad19088 cad19089 cad19094 aag02357 aan32980 aan32981 aaf26922 aas98781 aas98785 aan85523 cad89776 cad89777 bab12210 bab12211 bab12213 baa83992 aaf19813 aaf19814 aao64404 aao64405 aao64407 aao64403 aao64406 aav97870 aav97872 aav97875 aav97876 aav97877 aas47564 aas47557 aas47560 caf05651 caf05647 caf05648 aac69588 list of orfs containing methyltransferase domains in various experimentally characterized nrps/pks gene clusters. table also lists the number and type of mt domains in each of the orfs and results from pfam analysis using cdd search. threading analysis of mt domains since domain boundaries can be identified correctly by aligning the sequence of multi domain proteins with the 3d structures of the corresponding single domain proteins, we attempted to identify other proteins in pdb which are structurally similar to these mt domains of pks/nrps enzymes. however, the lack of crystal structures for any mt domains from pks/nrps biosynthetic pathway and high degree of sequence divergence in this enzyme family prompted us to use threading or fold recognition approach. these tools can potentially reveal structural similarity in absence of high degree of similarity in sequences. genthreader and phyre fold prediction servers were used for threading analysis. as discussed in the methods section, the mt sequence stretch identified by cdd or nrps-pks along with their flanking linkers was threaded on various structural folds in pdb. in cases where chemical structure of metabolite indicated presence of mt domains but no mt domain was detected by these programs, all linker stretches having unusual length were analyzed by both these fold recognition servers. column gives the unique name assigned to each mt containing sequence stretch, while the second column indicates their length. column 3– lists the pdb ids for the structures which show alignment with these sequences in fold recognition analysis using genthreader and phyre servers. results from genthreader corresponding to confidence level certain and high are labeled as c and h respectively. similarly, phyre results corresponding to precision level 100% and 95% are labeled as and respectively in curly braces. indicates the absence of that particular fold. it may be noted that the small molecule mt structures present in pdb show significant variation in their length and in case of some of these sequences alignments were found with mt structures having significant difference in length. for example, of the c-mt domains and n-mt domain in our data set aligned with aclacinomycin-10-hydroxylase and histamine n-mt. the huge length difference between these two mt structures made the choice of correct structural template a difficult task. 1qzz aligns with the beginning of the mt containing sequence stretch, while in the alignment of the same sequences with 1vlm, the length of the linker preceding the mt domain ranges from to even residues. however, careful manual analysis of the alignments as well as the corresponding structural templates indicated that, the crystal structure 1qzz consists of a predominantly α-helical n-terminal domain which is involved in dimerization and a c-terminal sam binding domain typical of methyltransferases. on the other hand, the crystal structure 1vlm contains the sam dependent mt domain alone. the overlapping region of both the alignments correspond to the catalytic domain of methyltransferase, while the linker sequences preceding the mt catalytic domain showed alignment with additional dimerization domains, if present in the structural template. earlier studies on structural analysis of small molecule methyltransferases have suggested that, these enzymes have a conserved sam-mt fold consisting of alternating β strands and α helices. earlier bioinformatics analysis of mt domains has also revealed that, despite high divergence in primary sequence, certain conserved sequence motifs are present in all sam dependent methyltransferases. therefore, we wanted to examine whether the conserved secondary structural elements and sequence motifs responsible for catalytic activity, substrate specificity and cofactor binding are conserved in the mt domains identified by threading alignments. figures and show the structure guided multiple alignment of representative n-mt, c-mt and o-mt sequences with the structural template 1vlm. as can be seen from figure the n-mt sequences have the conserved motifs i, ii/y, iv and v in n-mts. similarly motifs i, motif i-post, ii and iii are present in the c-mt and o-mt sequences. thus all the motifs identified in earlier analysis of small molecule methyltransferase sequences were found to be conserved in the multiple sequence alignments of c-, o- and n-mt domains identified by our threading analysis. the average percent identity among the c-, o- and n-mt domains was found to be and 17% respectively. the threading analysis also showed statistically significant matches with proteins other than methyltransferases. such matches were specifically seen for mt containing sequences which were longer in length due to the presence of large flanking linker sequences. as can be seen from table and figure the n-terminal region of mt containing sequence from bleomycin showed an alignment with the last amino acids of the ks-at di-domain structure from erythromycin pks. this stretch corresponds to the last helix of the at domain and a segment of the at-dh linker region. the c-terminal amino acid stretch of the mt containing sequence from bleomycin, nodularin and melithiazol also showed highly significant alignments with the structural subdomain of the recently elucidated structure of kr from erythromycin pks. these results suggest that, in type i pks proteins, the linker sequences preceding the mt domain i.e. at-mt linkers are homologous to the at-dh linkers and mt-kr linker regions are likely to adopt a short chain reductase fold and would constitute the structural half of the kr domain as demonstrated in erythromycin pks. thus mt catalytic domain is likely to be amino acids only. development of a computational protocol for identifying mt domains and their classification as c-mt, o-mt and n-mt the domain boundaries were thus identified with 1vlm and 1vl as templates and the regions which aligned with these templates were extracted from the mt domain sequences. they represented the curated mt domains with correct boundary. a set of mt domains out of these sequences were used as test set to check if mt domains can be correctly classified by pairwise alignment with these template sequences. each of the mt domains was queried against mt templates to find the number of mt domains identified by these templates. the query mt domain was classified as c-mt, o-mt or n-mt depending on the highest scoring match from the template set. the results obtained by the pairwise comparison of query sequences with the template sequences indicated that c-mt templates were able to identify all other c-mt sequences. however, there were a few o- and n-mts, which were also recognized by these c-mt templates. specifically, mt sequences in onnamide gene cluster showed very high similarity to c-mt while they are functionally annotated as o-mts by the authors who reported experimental characterization of this gene cluster. these o-mts also have motif i as exgxg which is characteristic of c-mts. an n-mt from pyochelin synthetase also exhibited considerable similarity to several c-mts and the two onnamide o-mts. in view of this apparent anomaly in sequence similarity of these proteins, their functional assignment needs to be examined carefully. in the orf apdb of anabaenopeptilide gene cluster, there are two mt-domains wherein the first one is entirely different from all the other mt sequences and does not show similarity with any other mt sequence. a dendrogram of all the mt sequences also illustrates the same pattern of results as obtained by these pairwise alignments. the two onnamide o-mts in onnb and onni genes and the n-mts in pyochelin and anabaenopeptilide show clustering with c-methyltransferases. a stand alone o-mt in stigmatellin is sequentially different from other o-methyltransferases, which was observed from the pairwise alignment results and is evident from the dendrogram. thus it can be concluded from the above analysis that these sequences can be used as templates to identify mt-domains in any given query sequence by pairwise alignment. these new mt templates were included in the current version of nrps-pks program for correct identification of various types of mt domains. profile hmms of c-, o- and n-methyltransferases an alternative approach to detect mt domains in a new sequence is to query that sequence against a database of hmm profiles. individual profiles were built for c-mt, o-mt and n-mts using the curated sequences. these profiles were then used to make a hmm profile database for mt domains. the set of c-mt, o-mt and n-mt sequences from experimentally characterized nrps/pks clusters were queried against this hmm database and the location of the mt-domain and their class was predicted in these sequences. table lists the score and e-value for alignment of representative mt sequences with the hmm profiles of n-mt, c-mt and o-mt domains. as can be seen from table most c-mt sequences show statistically significant alignment with o-mt profiles and vice versa. on the other hand, only the n-mt sequences from pyochelin aligned with the n-mt as well as c-mt profiles unlike the other n-mt sequences which aligned with n-mt profile alone. this finding is consistent with results from pairwise sequence alignment discussed in the previous section. a'-'sign indicates that alignments resulted in scores with e-value higher than e- several c-mts align with o-mt profiles, while only n-mt of pyochelin synthase shows alignment with c-mt profile as well. in order to test the predictive ability of our mt hmm profiles further, the recent version of the nr database of ncbi was also searched using these profiles to identify putative nrps/pks mt domains in various proteins. the sequences which matched with these profiles were grouped as c-mt, o-mt and n-mt containing sequences based on highest scoring profile match. the complete domain architecture of these mt containing proteins were also analyzed in details. the nr database search identified stand alone mt proteins and multifunctional proteins containing mt domains. out of the stand alone mt proteins, were o-mt proteins. in contrast to the very large number of stand alone o-mt proteins, there were only stand alone c-mt and stand alone n-mt proteins. even though experimentally characterized nrps/pks biosynthetic pathways have a relatively larger number of stand alone o-mt proteins compared to stand alone c-mt or n-mt domains, it is not apparent whether all these stand alone o-mt proteins identified by our profile search would indeed be associated with secondary metabolite biosynthesis. analysis of domain architectures in multifunctional proteins containing mt domains revealed several interesting results. these mt containing multifunctional proteins can be divided into four major groups. they are proteins containing mt domains along with core pks domains, core nrps domains, pks as well nrps domains and other catalytic domains. figure shows the number of proteins containing n-mt, o-mt and c-mt domains for each of the four categories. as can be seen from figure 10a, pks proteins have c-mt domains, pks proteins have o-mt and none of them have n-mt domains. this result is consistent with the fact that polyketides have no sites for n-methylation and o-methylation in known pks biosynthetic pathways is catalyzed by stand alone o-mts. this indicates that our profiles are able to correctly classify the three different classes of secondary metabolite methyltransferases. our nr database search also identified nrps proteins containing n-mt domains, containing o-mt domains and containing c-mt domains. since most nonribosomal peptide products are n-methylated, the presence of such a large number of c-mt domains as c-a-cmt-t modules was surprising. similarly, the hybrid nrps/pks set also had proteins containing c-mt domains as compared to only proteins containing n-mt domains. in fact of these c-mt domains in hybrid nrps/pks proteins were present next to condensation domains of nrps as c-cmt-pcp modules, while one would a priori expect n-mt domains in such modules instead of c-mt domains. in view of this finding of anomalously large number of nrps modules containing c-mt domains in nrps and hybrid nrps/pks proteins, we decided to analyze these proteins by pairwise alignment with representative mt templates from known nrps/pks biosynthetic pathways. interestingly the n-mt domain of pyochelin synthase was found to be closest match for out of the c-mt containing nrps proteins identified by profile search. most of these nrps proteins showed very high percentage identity with pyochelin synthase n-mt ranging from 27% to 69%. thus it is very much likely that, these nrps proteins indeed contain pyochelin type n-mt domains which are different from other n-mt domains. they were annotated by profile approach as c-mts, because n-mt domain of pyochelin synthase shows homology to c-mts and has comparable scores with c-mt as well as n-mt profiles. similarly, a c-mt domain from yersiniabactin synthase was found to be the closest homolog of c-mts found in nrps modules of hybrid nrps/pks proteins and the sequence similarity was also very high. this mt domain in yersiniabactin synthase catalyzes c-methylation, but is present as c-cmt-pcp module similar to the domain organization found in c-mt containing hybrid nrps/pks proteins found by our profile search. this suggests that our profile search has genuinely identified yersiniabactin type novel c-mt domains embedded in nrps modules. in case of other nrps proteins which showed matches with c-mt profiles, the c-mt domains from yersiniabactin, leinamycin and nodularin were found to be closest match. table shows the domain organization predicted for each of them by hmm profiles, and the percentage identity and similarity with closest matching mt domain in our representative templates set. even though the closest homolog approach also detects c-mt domains in these proteins in agreement with results from hmm approach, in view of the relatively low sequence identity with c-mts from known nrps/pks biosynthetic pathways, it is not clear if these domains are likely to be genuine c-mt domains as in yersiniabactin synthase or a different class of n-mt domains which lack homology to known n-mt domains in our data set. thus our analysis of nr database has revealed presence of many c-mt domains in nrps modules as in yersiniabactin. it has also identified several pyochelin type n-mt domains which often show higher homology to c-mt profiles. none of these proteins are currently experimentally characterized. experimental characterization of some of these mt domains would help in understanding how mt domains in nrps/pks family have evolved to acquire specificities for different substrates. however, a close examination of the chemical structures of pyochelin and yersiniabactin provides a rational for presence of n-mt domains with homology to c-mt sequences. as can be seen from figure in both these biosynthetic clusters mt domains transfer methyl groups to a five membered rings having very similar chemical structures. they only differ by the site of methylation. in view of the similarity in the structure of the acceptor substrate, pyochelin type n-mt domains show homology to c-mt proteins. it may be interesting to note that, in a recent study involving mts from type ii pks pathways, hertweck and coworkers have observed that, these type ii mts cluster according to the site of methylation not necessarily as c-mts, o-mts and n-mts. based on this observation, they have proposed that for polyketide alkylation, regioselectivity is more dominant than the type of nucleophile that is being alkylated. thus the c-mt domains found in nrps modules by our profile search could indeed be due to such correlation between sequence of type i mts and their regioselectivity. these observations have interesting implications of prediction of chemical structures of metabolites by genome mining. list of protein sequences from nr database which contain c-mt domains adjacent to the core nrps domains as identified by our mt hmm profile search. gene identifier number, domain organization and organism name is listed in columns and columns and list the closest match with the representative mt domains from experimentally characterized nrps/pks clusters and the corresponding percentage identities. the analysis of mt containing proteins in nr database also revealed the co-occurrence of nrps/pks type mt domains with several other catalytic domains apart from core pks and nrps domains. however, in most cases these proteins had o-mt domains and there were relatively few c-mt and n-mt domains. table shows domain organization for representative proteins from each category and also lists the number of such proteins identified by our hmm profile search. as can be seen, the o-mt domains are present along with a variety of catalytic domains which do not belong to core nrps or pks family. interestingly out of the examples correspond to co-occurrence with glycosyltransferase domains. since glycosyltransferases are a major class of tailoring enzymes involved in secondary metabolite biosynthesis, many of these proteins might indeed be associated with novel pks/nrps biosynthetic pathways. apart from glycosyltransferases, the other functional domains which occur with o-mt are oxidases, hydroxylases, tetratricopeptide repeat, phosphodiasterases, dna binding helix-turn helix proteins etc. it would be necessary to further analyze each of these proteins in details to understand their biochemical function. in contrast to o-mts, the n-mt and c-mt domains are present in multifunctional enzymes which contain other catalytic domains in addition to pks and nrps domains. some of the interesting examples are nrps/pks proteins containing chalcone synthase or carnitine acyltransferase domains along with c-mt domains. similarly, n-mt domains are present along with domains associated with streptococcal surface antigen, bacterial luciferase, phosphoenol pyruvate utilizing enzyme and ketopantoate reductase etc. thus these results give valuable clues about organisms which can potentially make secondary metabolites with a variety of structural modifications by diverse types of tailoring enzymes. some of these proteins would be interesting targets for detailed experimental investigation. list of representative protein sequences from nr database which contain c-mt, n-mt or o-mt domains in combination with functional domains other than core pks or nrps domains. gene identifier number, domain organization and organism names are listed in columns and respectively, while column lists total number of proteins having similar domain organization. discussions we have carried out a comprehensive analysis of the sequence and structural features of the mt domains present in multi functional proteins encoded by various experimentally characterized nrps, pks and hybrid nrps/pks gene clusters with known secondary metabolite products. even though presence of methyltransferase domains in nrps and pks family of megasynthases have been inferred from methylation pattern of the chemical structure of the secondary metabolite product, earlier studies have not defined the correct domain boundaries. threading analysis of the mt containing sequence stretches suggest 1vlm and 1vl as possible structural templates for nrps/pks mt domains. based on the alignment with these structural templates, we identify the correct boundaries and predict that, the general length of mt domains will be in the range of 200– residues. our threading analysis also reveals interesting homology between at-mt and at-dh linkers. similarly, large sequence stretches c-terminus to the c-mt domains of pks proteins are found to have homology with structural half of kr domains. these results not only explain the large variation in the length of the mt containing sequence stretch, they can also provide valuable clues for design of domain swapping experiments. the curated sequences of these mt domains were further analyzed. from the mt sequences of correct length, a representative set of mt domains covering the entire range of sequence divergence were chosen. a novel protocol for identification of mt domains by pairwise alignment and their classification as n-mt, c-mt and o-mt was developed using these mts as multiple templates. this mt domain identification protocol has been implemented in the current version of the program nrps-pks. using this approach c-mt domains were annotated in the pks proteins of dictyostelium discoideum in a recent work. the mt sequences with correct domain boundaries were also used to build profile hmms for o-mt, c-mt and n-mt domains. using these hmm profiles searches were carried out in the nr database of ncbi for identifying various other proteins containing c-mt, o-mt and n-mt domains. it is interesting to note that, apart from core pks and nrps domains, these secondary metabolite biosynthetic mt domains are also associated with other important catalytic domains like glycosyltransferase, oxidases, hydroxylases, phosphodiesterases and reductases. these proteins could be interesting targets for experimental characterization. our analysis also surprisingly revealed the presence of a large number c-mt domains in nrps modules adjacent to condensation and adenylation domains. these predicted c-mt domains can be classified into two groups. one group of c-mt domains showed high homology to n-mt domain of pyochelin synthase, which is different from typical n-mt domains present in nrps modules. unlike typical n-mt domains of nrps proteins, this shows homology to c-mt domains but catalyzes transfer of methyl group to nitrogen. this group could indeed be pyochelin type n-mt domains, but are mis-classified as c-mt due to their homology with c-mt proteins and under representation of pyochelin type n-mt in our training data set. the closest homolog of the other group of c-mt domains present in nrps modules is the c-mt domain of yersiniabactin synthase, which is present adjacent to a condensation domain of nrps, but is indeed a c-mt. however, in view of the low homology between the c-mt from yersiniabactin and this second group of predicted c-mts, it is difficult to predict whether they are yersiniabactin or pyochelin type mts. a close examination of the chemical structures of the yersiniabactin and pyochelin provides an evolutionary basis for the presence of pyochelin type n-mt domains having homology with c-mt proteins. as can be seen from figure an identical five membered ring is the acceptor moiety for these two classes of mts, while pyochelin n-mt methylates at the n position of the ring, the yersiniabactin c-mt methylates the adjacent c position. it may be interesting to note that such correlation between regioselectivity of the site of methylation and mt sequence have also been reported earlier by hertweck and coworkers for mts in type ii pks biosynthetic pathways. experimental characterization of proteins identified by our analysis would help in building more specific profiles for these novel mt domains.  we have carried out a comprehensive bioinformatics analysis of methyltransferase domains present in pks/nrps clusters having known secondary metabolite products. based on the site of methylation of these known secondary metabolites, the mt domains have been grouped as n-mt, c-mt and o-mt proteins and sequence/structural features have been analyzed in detail for each group. based on the results of this analysis, we have developed a novel knowledge based computational approach for detecting mt domains present in pks and nrps megasynthases, delineating their correct boundaries and classifying them as n-mt, c-mt and o-mt using profile hmms. analysis of proteins in nr database of ncbi using these class specific profiles has revealed several interesting examples of mt domains with novel substrate specificities. our analysis has also given interesting insight into the evolutionary basis of the novel substrate specificities of these mt proteins. these results have interesting implications for identification of novel secondary metabolites by genome mining and also rational design of novel natural products by biosynthetic engineering. 
a number of different tailoring enzymes catalyze a variety of chemical modifications to the polyketide or nonribosomal peptide backbone of these secondary metabolites to enhance their structural diversity. based on the results of this analysis, we have developed a novel knowledge based computational approach for detecting mt domains present in pks and nrps megasynthases, delineating their correct boundaries and classifying them as n-mt, c-mt and o-mt using profile hmms. in this work, we have carried out a comprehensive bioinformatics analysis of methyltransferase domains present in multi functional type i pks and nrps proteins encoded by pks/nrps gene clusters having known secondary metabolite products. analysis of nr database using this approach has revealed presence of several novel mt domains. analysis of proteins in nr database of ncbi using these class specific profiles has revealed several interesting examples, namely, c-mt domains in nrps modules, n-mt domains with significant homology to c-mt proteins, and presence of nrps/pks mts in association with other catalytic domains. these two classes of mts recognize similar acceptor substrates, but transfer methyl groups to n and c positions on these substrates. our analysis of the chemical structures of the secondary metabolites and their site of methylation suggested that a possible evolutionary basis for the presence of a novel class of n-mt domains with significant homology to c-mt proteins could be the close resemblance of the chemical structures of the acceptor substrates, as in the case of pyochelin and yersiniabactin. our analysis has also given interesting insight into the evolutionary basis of the novel substrate specificities of these mt proteins. therefore, development of powerful bioinformatics methods for identification of these tailoring enzymes and assignment of their substrate specificity is crucial for deciphering novel secondary metabolites by genome mining. we have developed a novel knowledge based computational approach for identifying mt domains present in type i pks and nrps multifunctional enzymes and predicting their site of methylation. secondary metabolites biosynthesized by polyketide synthase and nonribosomal peptide synthetase family of enzymes constitute several classes of therapeutically important natural products like erythromycin, rapamycin, cyclosporine etc. in view of their relevance for natural product based drug discovery, identification of novel secondary metabolite natural products by genome mining has been an area of active research.
 secondary metabolites biosynthesized by polyketide synthase and nonribosomal peptide synthetase family of enzymes constitute several classes of therapeutically important natural products like erythromycin, rapamycin, cyclosporine etc. in view of their relevance for natural product based drug discovery, identification of novel secondary metabolite natural products by genome mining has been an area of active research. a number of different tailoring enzymes catalyze a variety of chemical modifications to the polyketide or nonribosomal peptide backbone of these secondary metabolites to enhance their structural diversity. therefore, development of powerful bioinformatics methods for identification of these tailoring enzymes and assignment of their substrate specificity is crucial for deciphering novel secondary metabolites by genome mining.  in this work, we have carried out a comprehensive bioinformatics analysis of methyltransferase domains present in multi functional type i pks and nrps proteins encoded by pks/nrps gene clusters having known secondary metabolite products. based on the results of this analysis, we have developed a novel knowledge based computational approach for detecting mt domains present in pks and nrps megasynthases, delineating their correct boundaries and classifying them as n-mt, c-mt and o-mt using profile hmms. analysis of proteins in nr database of ncbi using these class specific profiles has revealed several interesting examples, namely, c-mt domains in nrps modules, n-mt domains with significant homology to c-mt proteins, and presence of nrps/pks mts in association with other catalytic domains. our analysis of the chemical structures of the secondary metabolites and their site of methylation suggested that a possible evolutionary basis for the presence of a novel class of n-mt domains with significant homology to c-mt proteins could be the close resemblance of the chemical structures of the acceptor substrates, as in the case of pyochelin and yersiniabactin. these two classes of mts recognize similar acceptor substrates, but transfer methyl groups to n and c positions on these substrates.  we have developed a novel knowledge based computational approach for identifying mt domains present in type i pks and nrps multifunctional enzymes and predicting their site of methylation. analysis of nr database using this approach has revealed presence of several novel mt domains. our analysis has also given interesting insight into the evolutionary basis of the novel substrate specificities of these mt proteins. 