BACKGROUND
klebsiella pneumoniae is a gram-negative, rod-shaped bacterium that is widely distributed in soil and water  <cit>  as well as the intestine, urethra and respiratory tract of mankind and other animals  <cit> .
this opportunistic pathogen has been regarded as one of the major causes of respiratory and urinary tract infections, septicemia and the third-most-common bacterial cause of hospital-acquired pneumonia in immunocompromised patients  <cit> .
studies in taiwan showed that this pathogen has the capacity to cause pyogenic liver abscess in human  <cit> .
similar cases have been observed in other countries as well, indicating that such medical infliction is not only confined to taiwan per se and may potentially emerge as a global problem  <cit> .
to add to this problem, k. pneumoniae strains which produce extended-spectrum beta-lactamases and are highly resistant to a spectrum of antibiotics are emerging worldwide  <cit> .
these strains, also known as k. pneumoniae carbapenemases -encoding strains, are often associated with nearly complete antibiotic resistance whereby failure and mortality rates related to pneumonia caused by this pathogen can reach up to 50% even with antibiotic therapy  <cit> .
this makes treatments for this bacterium more difficult and has certainly created obstacles, no less danger, to public health.
many components of the bacteria have been identified as pathogenic factors such as its capsular polysaccharide  <cit> , yersiniabactin  <cit>  and enterobactin  <cit> .
all these pathogenic factors are well characterized in which their mechanisms of action and effects are established.
little is known, however, on the roles of poorly characterized biomolecules such as hypothetical proteins  of this pathogen.
as their sequences and structures remain largely non-similar with other known proteins, hps are often regarded as proteins of unknown functions  <cit>  or orphan proteins  <cit> .
it is important to note that a substantial fraction  of any sequenced bacterial genomes consist of genes which encode hps  <cit> .
this is certainly the case even in model organisms such as escherichia coli, bacillus subtilis or saccharomyces cerevisiae <cit> .
efforts to gain basic understanding on the roles and possible functions of hps are crucial to close the gap between the “knowns” and the “unknowns”.
this is especially important to fit and complete the genetic information puzzle of any living organisms, as well as to gain a ‘complete’ understanding of these organisms as biological systems as a whole  <cit> .
as 25% of the complete genome sequence of k. pneumoniae mgh  <dig> codes for hps  <cit> , it serves as a good mining pool for these proteins to be studied structurally and functionally.
this effort is important particularly in substantiating the biological role and importance of hps in the system of a pathogen.
improved understanding of these proteins may make them potential targets of antimicrobial drugs  <cit> .
this present study highlights the in silico studies to characterize a hp, kpn_ <dig>  from k. pneumoniae mgh  <dig>  the results revealed that kpn_ <dig> is a zn-metalloprotease possibly related to the functions of the cell wall whereby its induced expression has interestingly changed the surface morphology of a lipoprotein-deficient e. coli je <dig> strain.
RESULTS
sequence analysis
the genome of k. pneumoniae mgh  <dig> was obtained from ncbi website  and thoroughly studied to identify the annotated proteins and hps.
a total of  <dig> hps were found in the genome of k. pneumoniae mgh  <dig>  via pblast analysis  <cit>  of the hps against the non-redundant  database, a particular hypothetical protein annotated as kpn_ <dig>  gave more than  <dig> hits with values above the e-value threshold of  <dig> .
majority of the top hits for this hp were also hps and proteins with unknown functions .
among these hits, a hypothetical lipoprotein from vibrio furnissii nctc  <dig> and twin-arginine translocation pathway signal peptide showed high similarity to kpn_ <dig>  up to 81%  and 99%, respectively .
a search on the structures of these top hits in the protein data bank  however, did not yield any result.
in other words, no potential structural template among these top hits was found.
conserved domains search on kpn_ <dig> using uniprot  <cit>  revealed that it contains a conserved domain found in the superfamily of m15_ <dig> peptidases.
using this information, a similarity search was performed on kpn_ <dig> against all peptidases in the merops peptidase database  <cit> .
blast merops results indicated that kpn_ <dig> shares similarity with many sub family m15a unassigned peptidases.
this family of peptidase consists of metallopeptidases mostly specialized carboxypeptidases and dipeptidases such as zn d-alanyl-d-alanine  carboxypeptidases.
the biological functions of d-ala-d-ala carboxypeptidases are related to bacterial cell wall biosynthesis and metabolism  <cit> .
to predict the possible function of kpn_ <dig>  recently reported protein prediction methods comprising of ffpred  <cit> , gostruct  <cit> , argot <dig>  <cit> , combfunc  <cit>  and pannzer  <cit>  were used.
out of this five prediction methods, gostruct and pannzer services were not available at the moment when the analyses were performed.
results obtained from ffpred indicated that kpn_ <dig> might be responsible for oxidation-reduction process with  <dig>  probability.
since ffpred system is dedicated to assign gene ontology terms for eukaryotic protein sequences  <cit> , the results obtained from the analysis of kpn_ <dig> using ffpred might not be accurate for a prokaryotic system.
analysis using combfunc and argot <dig> failed to predict any significant function for kpn_ <dig> although both highlighted that the protein consisted the peptidase m_ <dig> domain as well as a leucine rich domain.
kegg orthology  group analysis on kpn_ <dig> only annotated it as a hypothetical protein, unrelated to any ko group.
therefore, kpn_ <dig> could not be associated with any pathways based on the ko analysis.
a multiple sequence alignment  analysis of kpn_ <dig> with sequences from six other organisms containing similar domain from the blast result was performed using clustalw  <cit> .
<dig> residues from kpn_ <dig> were identical with these proteins .
there were also  <dig> residues  which are conserved suggesting that the same chemical properties are shared albeit differences in sequence identity.
two histidine residues i.e.
his <dig> and his <dig>  and an aspartate residue i.e.
asp <dig> which are postulated to be involved in zn chelation in well-characterized d-ala-d-ala carboxypeptidases, are interestingly found to be conserved here.
template selection
a search in the protein data bank  on the potential structural template to be used to build the model of kpn_ <dig> was performed.
no structure with high homology in pdb was detected for kpn_ <dig>  however, since the msa results confirmed the integrity of the putative peptidase_m15_ <dig> superfamily conserved domain in kpn_ <dig>  this domain was used to search for such potential template in pdb instead.
this resulted in the identification of one potential template, termed 1lbu with sequence identity of only 23%.
1lbu is a crystal structure of muramoyl-pentapeptide carboxypeptidase, a zn2+ d-ala-d-ala carboxypeptidase from streptomyces albus <cit>  which contain the particular peptidase_m15_ <dig> superfamily domain.
1lbu was also ranked top by phyre <dig> search within combfunc server as having the highest structural similarity kpn_ <dig>  the results indicated that kpn_ <dig> might adopt similar fold and domain with 1lbu; namely the hedgehog/d-ala-d-ala peptidase fold and zn2+ d-ala-d-ala carboxypeptidase c-terminal catalytic domain.
in fact, all the hits listed by phyre <dig> contained the hedgehog/d-ala-d-ala peptidase fold .
these results further stressed 1lbu as the best structural template for kpn_ <dig>
phylogenetics analysis via scop search  <cit>  was performed between kpn_ <dig> with other members of peptidase_m15_ <dig> superfamily to determine their degree of evolutionary relatedness.
the analysis revealed that 1lbu was at a further clad from kpn_ <dig>  in the cladrogram as compared to other organisms .
although the sequence identity of 1lbu compared with kpn_ <dig> is only 23%, it is evolutionary closer to klebsiella sp based on its phylogenetic relationship.
moreover, the length of 1lbu is similar to kpn_ <dig> .
thus, 1lbu was selected as the template for homology modeling.
homology modelling of kpn_ <dig> and model validation
homology modeling of kpn_ <dig> using modeller 9v <dig>  <cit>  with 1lbu as the template randomly generated  <dig> models.
the best model  was subsequently validated using procheck  <cit> .
the ramachandran analysis revealed that  <dig> % of the amino acid residues reside in the most favourable and additional allowed regions .
the built model was further verified using verify3d  <cit>  and errat  <cit> .
verify3d indicated that the built protein model scored  <dig> %, suggesting compatibility between the amino acid sequence and the environment of the amino acid side chains in the model.
errat analysis on the protein model gave forth score of  <dig> , a relatively acceptable assessment value on the arrangement of atoms with respect to one another in the protein model.
in addition to these analyses, the compactness of the built model was also validated using proq protein quality prediction tool  <cit> .
the result showed lg score of  <dig>  and maxsub score of  <dig> , indicating that the built model of kpn_ <dig> is within the range of an acceptable model.
calculations of the interaction energy and z-score using prosa-web  <cit>  energy plot for each residue of the model gave forth value of − <dig>  kcalmol- <dig>  based on these various structural evaluation results, the particular model can be accepted as a potential model for kpn_ <dig> .
structural and motif analyses
structural alignment of kpn_ <dig> with the template 1lbu and 2vo <dig>   <cit>  showed the integrity of the conserved domain.
structural analysis showed that the secondary structure, in particular the four beta stranded region and one single helix region, are well aligned .
this is a unique secondary structure topology shared among metalloproteases  <cit> .
the average rmsd between kpn_ <dig> and these two other structures is  <dig>  Å. further analysis on the secondary structure elements of the built model with 1lbu and 2vo <dig> using stride  <cit>  lends further support that these proteins share conserved secondary structure topologies .
certain peptidases, particularly those of peptidoglycan hydrolases such as d-ala-d-ala metallopeptidases are believed to contain a zn2+ ligand in most of the structures where the metal ion is coordinated by two histidines, an aspartate and a water molecule  <cit> .
the presence of these active site residues is clearly observed in our built model, where his <dig>  his <dig> and asp <dig> are located exactly at zn2+-chelating positions .
interestingly, these residues are also found to be highly conserved in certain hypothetical proteins from other organisms as well .
it has been reported that other than these active site residues, there is another second conserved his residue two residues upstream of the his zn2+ ligand  <cit> .
this particular his residue, his <dig>  was observed in the sequence of kpn_ <dig> where it is located two residues upstream of his <dig>  .
the intactness of both the secondary structure topology and the three zn2+ ligand-binding residues of the built model suggest that kpn_ <dig> may function as a cell wall -hydrolyzing enzyme, similar to a few characterized zn d-ala-d-ala metallopeptidases such as muramoyl-pentapeptide carboxypeptidase from s. albus <cit>  and vanx from enterococcus faecalis <cit> .
closer inspection on the sequence of kpn_ <dig> in comparison to the abovementioned proteins revealed that it does not contain the characteristic h-x-h motif which is predominantly found in nearly all d-ala-d-ala metallopeptidases, except vanx  <cit> .
this motif is present in the sequence of 1lbu, the template used for the homology modeling of kpn_ <dig> .
in the case of vanx, instead of the signature h-x-h motif it bears the e-x-x-h motif in its sequence  <cit> .
this motif was absent also in the sequence of kpn_ <dig>  however, kpn_ <dig> was found to possess the h-x -d motif similar to mepa peptidase .
similar to the zn d-ala-d-ala metallopeptidases stated above, mepa is a zn-metalloprotein shown to be involved in cell wall related functions  <cit> .
the only deviation to this similarity is the absence of the h-x-h motif in kpn_ <dig>  which is reported to be present in mepa  <cit> .
amplification and cloning of kpn_00953
to characterize further the possible function of kpn_ <dig>  its open reading frame  was amplified from the genome of k. pneumoniae mgh  <dig> using specifically designed primers.
a specific amplicon of 657 bp was obtained ).
cloning of this amplicon into pgem®-7zf  was subsequently achieved, as confirmed from blue-white screening , colony pcr ) and sequencing .
altered cell surface morphology of e. coli je <dig> overexpressing kpn_00953
since homology modeling results point to the possibility of kpn_ <dig> having cell wall related metabolic functions i.e.
peptidoglycan degradation, the effect of overexpressing this hp on cell surface morphology was investigated.
for this purpose, the cloned kpn_ <dig> construct was introduced into the lipoprotein-deficient e. coli je <dig> strain  <cit>  and subsequently overexpressed via iptg induction.
cells which overexpressed kpn_ <dig> appeared to have different surface morphology than cells which do not expressed this protein.
they appear to be slightly smoother with flattened edges ), and some of them seem to have deposits on their surfaces  and ).
in contrast, the cells which contained only the expression vector  have more well-defined and rougher surface texture ).
such alterations and deposits observed on the surface of the cells may suggest possible cell wall degradation by kpn_ <dig>
discussion
we have identified that hp kpn_ <dig> from k. pneumoniae mgh  <dig> contains a well conserved domain belonging to the m <dig> superfamily of peptidases.
template identification based on this domain has led to the building of a 3d model of kpn_ <dig> via homology modeling using the crystal structure of muramoyl-pentapeptide carboxypeptidase , a zn d-ala-d-ala metallopeptidase from s. albus <cit>  as the template.
the built model has been verified to be acceptable and topologically conserved with other available structures related to peptidases such as the l-alanoyl-d-glutamate endopeptidase domain of listeria bacteriophage endolysin ply <dig>   <cit> .
two his residues, his <dig> and his <dig>  as well as an asp residue, asp <dig> of this model are postulated to be involved in zn chelation  and are interestingly found to be conserved in other well characterized zn-metalloproteases  <cit> .
it is important to note that several members of zn-metalloproteases act as peptidoglycan hydrolases; in which they are involved in cell wall metabolism.
the cell wall of bacteria contains peptidoglycan which is important in preserving and maintaining the structural integrity of the cell by withstanding turgor.
it is closely linked to several physiological processes such as cell growth and division.
inhibition of its biosynthesis via the action of antiobiotics for instance as well as its degradation by lysozyme will result in cell lysis  <cit> .
peptidoglycan in general is made of alternating units of n-acetyl-glucosamine and n-acetyl-muramic acid that are linked via  <dig> -glycosidic bonds.
the muramyl residues serve as platforms for the attachment of short polypeptides which contain both l- and d-amino acids and typically have two d-ala residues at the c terminus.
these peptide components on the muramyl residues can be crosslinked by transpeptidation, which subsequently will result in a loss of the terminal d-ala and strengthening of the bacterial cell wall  <cit> .
members of zn-metalloproteases which have been characterized to be involved in peptidoglycan  biosynthesis and metabolism include zn d-ala-d-ala carboxypeptidases and dipeptidases  <cit> .
muramoyl-pentapeptide carboxypeptidase, which is used as a template in the homology modeling of kpn_ <dig>  is a specific zn d-ala-d-ala carboxypeptidase  <cit> .
it was reported that this particular enzyme from streptomyces hydrolyzes the c-terminal peptide bond of peptides of general structure r-d-ala-d-xaa.
the lytic and extracellular characteristics of the enzyme brought about the suggestion that this enzyme is used by streptomyces for fighting competitors in its ecological niche since it does not hydrolyze the streptomyces peptidoglycan  <cit> .
another instance is the zn-dependent d-ala-d-ala  dipeptidase, vanx.
this peptidase reduces the cellular pool of the d-ala-d-ala dipeptide so that only the d-ala-d-lactate peptidoglycan chain precursors are produced and incorporated into the cell wall instead of the former.
the modified peptidoglycan reportedly exhibited a  <dig> -fold decrease in affinity for vancomycin.
this feature is responsible in conferring antibiotic resistance to pathogenic bacteria such as vancomycin-resistant enterococci   <cit> .
both of these proteins were reported to be similar and structurally related  <cit>  despite the differences in their sequences.
the muramoyl-pentapeptide carboxypeptidase from s. albus, like nearly all zn d-ala-d-ala metallopeptidases, contains the h-x-h motif.
vanx, on the other hand, contains the e-x-x-h motif making it an exception among the d-ala-d-ala metallopeptidases  <cit> .
in the case of kpn_ <dig>  it does not possess both of these characteristic motifs.
instead, it contains the h-x -d motif, similar to mepa peptidase  <cit> .
however, it is important to stress that mepa contains as well the characteristic h-x-h motif  <cit>  which kpn_ <dig> lacks.
in terms of molecular function, mepa is reported to cleave d-alanyl-meso- <dig> -diamino-pimelyl peptide bonds in e. coli peptidoglycan and is classified as a peptidase of unknown fold and catalytic class due to its low sequence similarity with other peptidases  <cit> .
kpn_ <dig> resembles mepa in this respect in which kpn_ <dig> is shown to be related to the subfamily m15a of unassigned peptidases from blast merops scan.
in contrast to mepa however, kpn_ <dig> could be assigned to a subfamily or catalytic class of peptidases, namely the m15a subfamily of peptidase.
as this subfamily of peptidases consists of a number of characterized members such as zn d-ala-d-ala carboxypeptidases, functional inference of kpn_ <dig> could be made based on this information.
although kpn_ <dig> contains the h-x -d motif of mepa, conserved domain analysis result seems to relate kpn_ <dig> more to zn d-ala-d-ala metallopeptidase in general, excluding vanx.
hence, the presence of the h-x -d motif of mepa in kpn_ <dig> may occur by chance.
it is important to note that whilst both domain analysis and homology modeling of kpn_ <dig> revealed the conservation of important domains, zn-chelating residues and secondary structure topologies to d-ala-d-ala carboxypeptidase, sequence similarity search revealed that this particular hp is also related to the twin-arginine translocation  pathway signal sequence.
tat pathway is a protein transport system for the export of folded proteins  <cit> .
proteins which are targeted to the tat pathway are exported to the cell envelope or to the extracellular space by tripartite n-terminal signal peptides and tat translocase, which are found in the cytoplasmic membrane  <cit> .
however, it is important to note that kpn_ <dig> lacks the consensus twin-arginine motif, -r-r-x-f-l-k, which is reported to be present in all types of bacterial signal peptides  <cit> , despite the sequence similarity mentioned above.
in addition to this, kpn_ <dig> does not contain signal peptide sequence as revealed by analyses using various signal peptide detection software such as signalp  <dig>   <cit> , signal–3 l  <cit> , ipsort  <cit>  and sosuisignal  <cit> .
hence, this omits the possibility that kpn_ <dig> is functionally related to tat pathway.
further inference on the possible function of kpn_ <dig> was attempted by cloning and expressing its gene in e. coli je <dig> strain which is deficient in lipoprotein production.
induced production of kpn_ <dig> in this particular strain changed the morphology of the cells.
they appeared to be smoother with less defined edges with some having deposits on their surfaces.
cells which did not produce the cloned kpn_ <dig> protein appeared to be rougher with defined edges .
this observation highlights the possibility of kpn_ <dig> to be involved in the functions of the cell wall, similar to other characterized peptidoglycan-hydrolyzing zn metallopeptidases.
CONCLUSIONS
based on the three-dimensional model, domain and residues conservation of kpn_ <dig> to d-ala-d-ala carboxypeptidase, we hypothesize that kpn_ <dig> adopts the functionality as a metallopeptidase with an important role in cell wall metabolism.
this is further supported by the altered surface morphology of e. coli je <dig> cells overexpressing kpn_ <dig>  the mechanism as to how kpn_ <dig> brings about these changes is worthy to be investigated in the near future.
this can be achieved via gene-knockout and structural biology studies to understand its structure, function and mechanism of action.
such efforts will undoubtedly address the possibility of utilizing this protein as a new drug target against k. pneumoniae in the future.

 klebsiella pneumoniae is a gram-negative, rod-shaped bacterium that is widely distributed in soil and water as well as the intestine, urethra and respiratory tract of mankind and other animals. this opportunistic pathogen has been regarded as one of the major causes of respiratory and urinary tract infections, septicemia and the third-most-common bacterial cause of hospital-acquired pneumonia in immunocompromised patients. studies in taiwan showed that this pathogen has the capacity to cause pyogenic liver abscess in human. similar cases have been observed in other countries as well, indicating that such medical infliction is not only confined to taiwan per se and may potentially emerge as a global problem. to add to this problem, k. pneumoniae strains which produce extended-spectrum beta-lactamases and are highly resistant to a spectrum of antibiotics are emerging worldwide. these strains, also known as k. pneumoniae carbapenemases -encoding strains, are often associated with nearly complete antibiotic resistance whereby failure and mortality rates related to pneumonia caused by this pathogen can reach up to 50% even with antibiotic therapy. this makes treatments for this bacterium more difficult and has certainly created obstacles, no less danger, to public health. many components of the bacteria have been identified as pathogenic factors such as its capsular polysaccharide, yersiniabactin and enterobactin. all these pathogenic factors are well characterized in which their mechanisms of action and effects are established. little is known, however, on the roles of poorly characterized biomolecules such as hypothetical proteins of this pathogen. as their sequences and structures remain largely non-similar with other known proteins, hps are often regarded as proteins of unknown functions or orphan proteins. it is important to note that a substantial fraction of any sequenced bacterial genomes consist of genes which encode hps. this is certainly the case even in model organisms such as escherichia coli, bacillus subtilis or saccharomyces cerevisiae. efforts to gain basic understanding on the roles and possible functions of hps are crucial to close the gap between the “knowns” and the “unknowns”. this is especially important to fit and complete the genetic information puzzle of any living organisms, as well as to gain a ‘complete’ understanding of these organisms as biological systems as a whole. as 25% of the complete genome sequence of k. pneumoniae mgh codes for hps, it serves as a good mining pool for these proteins to be studied structurally and functionally. this effort is important particularly in substantiating the biological role and importance of hps in the system of a pathogen. improved understanding of these proteins may make them potential targets of antimicrobial drugs. this present study highlights the in silico studies to characterize a hp, kpn_ from k. pneumoniae mgh the results revealed that kpn_ is a zn-metalloprotease possibly related to the functions of the cell wall whereby its induced expression has interestingly changed the surface morphology of a lipoprotein-deficient e. coli je strain.  sequence analysis the genome of k. pneumoniae mgh was obtained from ncbi website and thoroughly studied to identify the annotated proteins and hps. a total of hps were found in the genome of k. pneumoniae mgh via pblast analysis of the hps against the non-redundant database, a particular hypothetical protein annotated as kpn_ gave more than hits with values above the e-value threshold of. majority of the top hits for this hp were also hps and proteins with unknown functions. among these hits, a hypothetical lipoprotein from vibrio furnissii nctc and twin-arginine translocation pathway signal peptide showed high similarity to kpn_ up to 81% and 99%, respectively. a search on the structures of these top hits in the protein data bank however, did not yield any result. in other words, no potential structural template among these top hits was found. conserved domains search on kpn_ using uniprot revealed that it contains a conserved domain found in the superfamily of m15_ peptidases. using this information, a similarity search was performed on kpn_ against all peptidases in the merops peptidase database. blast merops results indicated that kpn_ shares similarity with many sub family m15a unassigned peptidases. this family of peptidase consists of metallopeptidases mostly specialized carboxypeptidases and dipeptidases such as zn d-alanyl-d-alanine carboxypeptidases. the biological functions of d-ala-d-ala carboxypeptidases are related to bacterial cell wall biosynthesis and metabolism. to predict the possible function of kpn_ recently reported protein prediction methods comprising of ffpred, gostruct, argot, combfunc and pannzer were used. out of this five prediction methods, gostruct and pannzer services were not available at the moment when the analyses were performed. results obtained from ffpred indicated that kpn_ might be responsible for oxidation-reduction process with probability. since ffpred system is dedicated to assign gene ontology terms for eukaryotic protein sequences, the results obtained from the analysis of kpn_ using ffpred might not be accurate for a prokaryotic system. analysis using combfunc and argot failed to predict any significant function for kpn_ although both highlighted that the protein consisted the peptidase m_ domain as well as a leucine rich domain. kegg orthology group analysis on kpn_ only annotated it as a hypothetical protein, unrelated to any ko group. therefore, kpn_ could not be associated with any pathways based on the ko analysis. a multiple sequence alignment analysis of kpn_ with sequences from six other organisms containing similar domain from the blast result was performed using clustalw. residues from kpn_ were identical with these proteins. there were also residues which are conserved suggesting that the same chemical properties are shared albeit differences in sequence identity. two histidine residues i.e. his and his and an aspartate residue i.e. asp which are postulated to be involved in zn chelation in well-characterized d-ala-d-ala carboxypeptidases, are interestingly found to be conserved here. template selection a search in the protein data bank on the potential structural template to be used to build the model of kpn_ was performed. no structure with high homology in pdb was detected for kpn_ however, since the msa results confirmed the integrity of the putative peptidase_m15_ superfamily conserved domain in kpn_ this domain was used to search for such potential template in pdb instead. this resulted in the identification of one potential template, termed 1lbu with sequence identity of only 23%. 1lbu is a crystal structure of muramoyl-pentapeptide carboxypeptidase, a zn2+ d-ala-d-ala carboxypeptidase from streptomyces albus which contain the particular peptidase_m15_ superfamily domain. 1lbu was also ranked top by phyre search within combfunc server as having the highest structural similarity kpn_ the results indicated that kpn_ might adopt similar fold and domain with 1lbu; namely the hedgehog/d-ala-d-ala peptidase fold and zn2+ d-ala-d-ala carboxypeptidase c-terminal catalytic domain. in fact, all the hits listed by phyre contained the hedgehog/d-ala-d-ala peptidase fold. these results further stressed 1lbu as the best structural template for kpn_ phylogenetics analysis via scop search was performed between kpn_ with other members of peptidase_m15_ superfamily to determine their degree of evolutionary relatedness. the analysis revealed that 1lbu was at a further clad from kpn_ in the cladrogram as compared to other organisms. although the sequence identity of 1lbu compared with kpn_ is only 23%, it is evolutionary closer to klebsiella sp based on its phylogenetic relationship. moreover, the length of 1lbu is similar to kpn_. thus, 1lbu was selected as the template for homology modeling. homology modelling of kpn_ and model validation homology modeling of kpn_ using modeller 9v with 1lbu as the template randomly generated models. the best model was subsequently validated using procheck. the ramachandran analysis revealed that % of the amino acid residues reside in the most favourable and additional allowed regions. the built model was further verified using verify3d and errat. verify3d indicated that the built protein model scored %, suggesting compatibility between the amino acid sequence and the environment of the amino acid side chains in the model. errat analysis on the protein model gave forth score of, a relatively acceptable assessment value on the arrangement of atoms with respect to one another in the protein model. in addition to these analyses, the compactness of the built model was also validated using proq protein quality prediction tool. the result showed lg score of and maxsub score of, indicating that the built model of kpn_ is within the range of an acceptable model. calculations of the interaction energy and z-score using prosa-web energy plot for each residue of the model gave forth value of − kcalmol- based on these various structural evaluation results, the particular model can be accepted as a potential model for kpn_. structural and motif analyses structural alignment of kpn_ with the template 1lbu and 2vo showed the integrity of the conserved domain. structural analysis showed that the secondary structure, in particular the four beta stranded region and one single helix region, are well aligned. this is a unique secondary structure topology shared among metalloproteases. the average rmsd between kpn_ and these two other structures is  Å. further analysis on the secondary structure elements of the built model with 1lbu and 2vo using stride lends further support that these proteins share conserved secondary structure topologies. certain peptidases, particularly those of peptidoglycan hydrolases such as d-ala-d-ala metallopeptidases are believed to contain a zn2+ ligand in most of the structures where the metal ion is coordinated by two histidines, an aspartate and a water molecule. the presence of these active site residues is clearly observed in our built model, where his his and asp are located exactly at zn2+-chelating positions. interestingly, these residues are also found to be highly conserved in certain hypothetical proteins from other organisms as well. it has been reported that other than these active site residues, there is another second conserved his residue two residues upstream of the his zn2+ ligand. this particular his residue, his was observed in the sequence of kpn_ where it is located two residues upstream of his. the intactness of both the secondary structure topology and the three zn2+ ligand-binding residues of the built model suggest that kpn_ may function as a cell wall -hydrolyzing enzyme, similar to a few characterized zn d-ala-d-ala metallopeptidases such as muramoyl-pentapeptide carboxypeptidase from s. albus and vanx from enterococcus faecalis. closer inspection on the sequence of kpn_ in comparison to the abovementioned proteins revealed that it does not contain the characteristic h-x-h motif which is predominantly found in nearly all d-ala-d-ala metallopeptidases, except vanx. this motif is present in the sequence of 1lbu, the template used for the homology modeling of kpn_. in the case of vanx, instead of the signature h-x-h motif it bears the e-x-x-h motif in its sequence. this motif was absent also in the sequence of kpn_ however, kpn_ was found to possess the h-x -d motif similar to mepa peptidase. similar to the zn d-ala-d-ala metallopeptidases stated above, mepa is a zn-metalloprotein shown to be involved in cell wall related functions. the only deviation to this similarity is the absence of the h-x-h motif in kpn_ which is reported to be present in mepa. amplification and cloning of kpn_00953 to characterize further the possible function of kpn_ its open reading frame was amplified from the genome of k. pneumoniae mgh using specifically designed primers. a specific amplicon of 657 bp was obtained ). cloning of this amplicon into pgem®-7zf was subsequently achieved, as confirmed from blue-white screening, colony pcr ) and sequencing. altered cell surface morphology of e. coli je overexpressing kpn_00953 since homology modeling results point to the possibility of kpn_ having cell wall related metabolic functions i.e. peptidoglycan degradation, the effect of overexpressing this hp on cell surface morphology was investigated. for this purpose, the cloned kpn_ construct was introduced into the lipoprotein-deficient e. coli je strain and subsequently overexpressed via iptg induction. cells which overexpressed kpn_ appeared to have different surface morphology than cells which do not expressed this protein. they appear to be slightly smoother with flattened edges ), and some of them seem to have deposits on their surfaces and ). in contrast, the cells which contained only the expression vector have more well-defined and rougher surface texture ). such alterations and deposits observed on the surface of the cells may suggest possible cell wall degradation by kpn_ discussion we have identified that hp kpn_ from k. pneumoniae mgh contains a well conserved domain belonging to the m superfamily of peptidases. template identification based on this domain has led to the building of a 3d model of kpn_ via homology modeling using the crystal structure of muramoyl-pentapeptide carboxypeptidase, a zn d-ala-d-ala metallopeptidase from s. albus as the template. the built model has been verified to be acceptable and topologically conserved with other available structures related to peptidases such as the l-alanoyl-d-glutamate endopeptidase domain of listeria bacteriophage endolysin ply. two his residues, his and his as well as an asp residue, asp of this model are postulated to be involved in zn chelation and are interestingly found to be conserved in other well characterized zn-metalloproteases. it is important to note that several members of zn-metalloproteases act as peptidoglycan hydrolases; in which they are involved in cell wall metabolism. the cell wall of bacteria contains peptidoglycan which is important in preserving and maintaining the structural integrity of the cell by withstanding turgor. it is closely linked to several physiological processes such as cell growth and division. inhibition of its biosynthesis via the action of antiobiotics for instance as well as its degradation by lysozyme will result in cell lysis. peptidoglycan in general is made of alternating units of n-acetyl-glucosamine and n-acetyl-muramic acid that are linked via -glycosidic bonds. the muramyl residues serve as platforms for the attachment of short polypeptides which contain both l- and d-amino acids and typically have two d-ala residues at the c terminus. these peptide components on the muramyl residues can be crosslinked by transpeptidation, which subsequently will result in a loss of the terminal d-ala and strengthening of the bacterial cell wall. members of zn-metalloproteases which have been characterized to be involved in peptidoglycan biosynthesis and metabolism include zn d-ala-d-ala carboxypeptidases and dipeptidases. muramoyl-pentapeptide carboxypeptidase, which is used as a template in the homology modeling of kpn_ is a specific zn d-ala-d-ala carboxypeptidase. it was reported that this particular enzyme from streptomyces hydrolyzes the c-terminal peptide bond of peptides of general structure r-d-ala-d-xaa. the lytic and extracellular characteristics of the enzyme brought about the suggestion that this enzyme is used by streptomyces for fighting competitors in its ecological niche since it does not hydrolyze the streptomyces peptidoglycan. another instance is the zn-dependent d-ala-d-ala dipeptidase, vanx. this peptidase reduces the cellular pool of the d-ala-d-ala dipeptide so that only the d-ala-d-lactate peptidoglycan chain precursors are produced and incorporated into the cell wall instead of the former. the modified peptidoglycan reportedly exhibited a -fold decrease in affinity for vancomycin. this feature is responsible in conferring antibiotic resistance to pathogenic bacteria such as vancomycin-resistant enterococci. both of these proteins were reported to be similar and structurally related despite the differences in their sequences. the muramoyl-pentapeptide carboxypeptidase from s. albus, like nearly all zn d-ala-d-ala metallopeptidases, contains the h-x-h motif. vanx, on the other hand, contains the e-x-x-h motif making it an exception among the d-ala-d-ala metallopeptidases. in the case of kpn_ it does not possess both of these characteristic motifs. instead, it contains the h-x -d motif, similar to mepa peptidase. however, it is important to stress that mepa contains as well the characteristic h-x-h motif which kpn_ lacks. in terms of molecular function, mepa is reported to cleave d-alanyl-meso- -diamino-pimelyl peptide bonds in e. coli peptidoglycan and is classified as a peptidase of unknown fold and catalytic class due to its low sequence similarity with other peptidases. kpn_ resembles mepa in this respect in which kpn_ is shown to be related to the subfamily m15a of unassigned peptidases from blast merops scan. in contrast to mepa however, kpn_ could be assigned to a subfamily or catalytic class of peptidases, namely the m15a subfamily of peptidase. as this subfamily of peptidases consists of a number of characterized members such as zn d-ala-d-ala carboxypeptidases, functional inference of kpn_ could be made based on this information. although kpn_ contains the h-x -d motif of mepa, conserved domain analysis result seems to relate kpn_ more to zn d-ala-d-ala metallopeptidase in general, excluding vanx. hence, the presence of the h-x -d motif of mepa in kpn_ may occur by chance. it is important to note that whilst both domain analysis and homology modeling of kpn_ revealed the conservation of important domains, zn-chelating residues and secondary structure topologies to d-ala-d-ala carboxypeptidase, sequence similarity search revealed that this particular hp is also related to the twin-arginine translocation pathway signal sequence. tat pathway is a protein transport system for the export of folded proteins. proteins which are targeted to the tat pathway are exported to the cell envelope or to the extracellular space by tripartite n-terminal signal peptides and tat translocase, which are found in the cytoplasmic membrane. however, it is important to note that kpn_ lacks the consensus twin-arginine motif, -r-r-x-f-l-k, which is reported to be present in all types of bacterial signal peptides, despite the sequence similarity mentioned above. in addition to this, kpn_ does not contain signal peptide sequence as revealed by analyses using various signal peptide detection software such as signalp, signal–3 l, ipsort and sosuisignal. hence, this omits the possibility that kpn_ is functionally related to tat pathway. further inference on the possible function of kpn_ was attempted by cloning and expressing its gene in e. coli je strain which is deficient in lipoprotein production. induced production of kpn_ in this particular strain changed the morphology of the cells. they appeared to be smoother with less defined edges with some having deposits on their surfaces. cells which did not produce the cloned kpn_ protein appeared to be rougher with defined edges. this observation highlights the possibility of kpn_ to be involved in the functions of the cell wall, similar to other characterized peptidoglycan-hydrolyzing zn metallopeptidases.  based on the three-dimensional model, domain and residues conservation of kpn_ to d-ala-d-ala carboxypeptidase, we hypothesize that kpn_ adopts the functionality as a metallopeptidase with an important role in cell wall metabolism. this is further supported by the altered surface morphology of e. coli je cells overexpressing kpn_ the mechanism as to how kpn_ brings about these changes is worthy to be investigated in the near future. this can be achieved via gene-knockout and structural biology studies to understand its structure, function and mechanism of action. such efforts will undoubtedly address the possibility of utilizing this protein as a new drug target against k. pneumoniae in the future. 
in the complete genome sequence of k. pneumoniae mgh approximately one-fourth of the genome encodes for hypothetical proteins. three-dimensional model of the protein showed that its secondary structure topology and active site are similar with those found among metalloproteases where two his residues, namely his and his and an asp residue, asp in kpn_ were found to be zn-chelating residues. as more k. pneumoniae strains are becoming highly resistant to various antibiotics, treatment of this bacterium has been rendered more difficult. medical inflictions by the pathogen can range from respiratory and urinary tract infections, septicemia and primarily, pneumonia. some members of this superfamily are metalloproteases which are involved in cell wall metabolism. as lipoproteins and other components of the cell wall are important pathogenic factors, homology modeling of kpn_ was attempted to predict the structure and function of this protein. we postulate that kpn_ is a zn metalloprotease and may play a role in bacterial cell wall metabolism. kpn_00953hypothetical proteinhomology modelingpeptidase m15_ superfamilycell wall metabolism structural biology studies to understand its structure, function and mechanism of action pose the possibility of utilizing this protein as a new drug target against k. pneumoniae in the future. klebsiella pneumoniae plays a major role in causing nosocomial infection in immunocompromised patients. blastp similarity search on kpn_ revealed that majority of the hits were hypothetical proteins although two of the hits suggested that it may be a lipoprotein or related to twin-arginine translocation pathway important for transport of proteins to the cell membrane and periplasmic space. this situation, as a consequence, poses a threat to public health. sequence analysis on the hps of k. pneumoniae mgh revealed that a particular hp termed kpn_ contains a m15_ peptidases superfamily conserved domain. due to their low homology and relatedness to other known proteins, hps may serve as potential, new drug targets.
 klebsiella pneumoniae plays a major role in causing nosocomial infection in immunocompromised patients. medical inflictions by the pathogen can range from respiratory and urinary tract infections, septicemia and primarily, pneumonia. as more k. pneumoniae strains are becoming highly resistant to various antibiotics, treatment of this bacterium has been rendered more difficult. this situation, as a consequence, poses a threat to public health. hence, identification of possible novel drug targets against this opportunistic pathogen need to be undertaken. in the complete genome sequence of k. pneumoniae mgh approximately one-fourth of the genome encodes for hypothetical proteins. due to their low homology and relatedness to other known proteins, hps may serve as potential, new drug targets.  sequence analysis on the hps of k. pneumoniae mgh revealed that a particular hp termed kpn_ contains a m15_ peptidases superfamily conserved domain. some members of this superfamily are metalloproteases which are involved in cell wall metabolism. blastp similarity search on kpn_ revealed that majority of the hits were hypothetical proteins although two of the hits suggested that it may be a lipoprotein or related to twin-arginine translocation pathway important for transport of proteins to the cell membrane and periplasmic space. as lipoproteins and other components of the cell wall are important pathogenic factors, homology modeling of kpn_ was attempted to predict the structure and function of this protein. three-dimensional model of the protein showed that its secondary structure topology and active site are similar with those found among metalloproteases where two his residues, namely his and his and an asp residue, asp in kpn_ were found to be zn-chelating residues. interestingly, induced expression of the cloned kpn_ gene in lipoprotein-deficient e. coli je resulted in smoother cells with flattened edges. some cells showed deposits of film-like material under scanning electron microscope.  we postulate that kpn_ is a zn metalloprotease and may play a role in bacterial cell wall metabolism. structural biology studies to understand its structure, function and mechanism of action pose the possibility of utilizing this protein as a new drug target against k. pneumoniae in the future. kpn_00953hypothetical proteinhomology modelingpeptidase m15_ superfamilycell wall metabolism 