Abstract:
The present invention relates to mutant 1,3-propanediol dehydrogenase and a novel microorganism that is capable of growing in concentrations of at least 105 g/l 1,3-propanediol, levels normally toxic to wild-type microorganisms. The present invention also provides expression vectors and host cells comprising the mutant 1,3-propanediol dehydrogenase as well as methods for producing 1,3-propanediol comprising the use of cells comprising the mutant 1,3-propanediol dehydrogenase.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention relates to mutant 1,3-propanediol dehydrogenase having an altered Km for 1,3-propanediol. The present invention provides the nucleic acid and amino acid sequence of the mutant form of 1,3-propanediol dehydrogenase. The present invention also provides expression vectors and host cells comprising mutant 1,3-propanediol dehydrogenase.  
         BACKGROUND OF THE INVENTION  
         [0002]    1,3-Propanediol is a monomer having potential utility in the production of polyester fibers and the manufacture of polyurethanes and cyclic compounds. The production of 1,3-propanediol has been disclosed in U.S. Pat. No. 5,686,276 issued Nov. 11, 1997 and WO 98/21341. One representative pathway for the production of 1,3-propanediol from glucose can be accomplished by the following series of steps. Glucose is converted in a series of steps by enzymes of the glycolytic pathway to dihydroxyacetone phosphate (DHAP) and 3-phosphoglyceraldehyde (3-PG). Glycerol is then formed by either hydrolysis of DHAP to dihydroxyacetone (DHA) followed by reduction, or reduction of DHAP to glycerol 3-phosphate (G3P) followed by hydrolysis. The hydrolysis step can be catalyzed by any number of cellular phosphatases which are known to be specific or non-specific with respect to their substrates or the activity can be introduced into the host by recombination. The reduction step can be catalyzed by a NAD +  (or NADP + ) linked host enzyme or the activity can be introduced into the host by recombination. It is notable that the dha regulon contains a glycerol dehydrogenase (E.C. 1.1.1.6) which catalyzes the reversible reaction of Equation 3. 
           Glycerol® 3-HP+H 2 O  (Equation 1) 
           3-HP+NADH+H + ® 1,3-Propanediol+NAD +   (Equation 2) 
           Glycerol+NAD + ® DHA+NADH+H +   (Equation 3) 
           [0003]    Glycerol is converted to 1,3-propanediol via the intermediate 3-hydroxypropionaldehye (3-HP) as has been described in detail above. The intermediate 3-HP is produced from glycerol (Equation 1) by a dehydratase enzyme which can be encoded by the host or can introduced into the host by recombination. This dehydratase can be glycerol dehydratase (E.C. 4.2.1.30), diol dehydratase (E.C. 4.2.1.28), or any other enzyme able to catalyze this transformation. Glycerol dehydratase is encoded by the dha regulon. 1,3-Propanediol is produced from 3-HP (Equation 2) by a NAD + -(or NADP + ) linked host enzyme or the activity can introduced into the host by recombination. This final reaction in the production of 1,3-propanediol can be catalyzed by 1,3-propanediol dehydrogenase (E.C. 1.1.1.202) or other alcohol dehydrogenases.  
           [0004]    In  Klebsiella pneumoniae  and  Citrobacter freundii,  the genes encoding the functionally linked activities of glycerol dehydratase (dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK) are encompassed by the dha regulon. The dha regulons from Citrobacter and Klebsiella have been expressed in  Escherichia coli  and have been shown to convert glycerol to 1,3-propanediol.  
           [0005]    Nucleic acid and amino acid sequences for a 1,3-propanediol dehydrogenase that have been disclosed in the art, including  Klebsiella pneumoniae  GenBank accession # U30903 (Williard, 1994, “Investigation of the  Klebsiella pneumoniae  1,3-propanediol pathway: Characterization and expression of glycerol dehydratase and 1,3-propanediol oxidoreductase” Thesis Chemical Engineering, University of Wisconsin-Madison);  Citrobacter freundii  GenBank accession # U09771 (Daniel, R. et al., 1995, Purification of 1,3-propanediol dehydrogenase from  Citrobacter freundii:  cloning, sequencing, and overexpression of the corresponding gene in  Escherichia coli. J. Bacteriol.  177:2151-2156); and  Clostridium pasteurianum  GenBank accession # AF006034 (Luers,F. et al., 1997, Glycerol conversion to 1,3-propanediol by  Clostridium pasteurianum:  cloning and expression of the gene encoding 1,3-propanediol dehydrogenase.  FEMS Microbiol. Lett.  154:337-345).  
         SUMMARY OF THE INVENTION  
         [0006]    The present invention relates to the discovery of a mutant form of 1,3-propanediol dehydrogenase (PDD) isolated from a derivative of  E.blattae  capable of growth in the presence of at least 105 g/l 1,3-propanediol, levels normally toxic to wild-type  E.blattae.  The present invention is therefore based in part upon the discovery that the mutant form of PDD is associated with  E.blattae&#39; s resistance to normally toxic levels of 1,3-propanediol. The present invention is also based in part upon the finding that this mutant PDD has an altered Km for 1,3-propanediol and NAD.  
           [0007]    Accordingly, the present invention provides a mutant PDD having a Km for 1,3-propanediol that is increased over the wild-type PDD Km for 1,3-propanediol. In one embodiment, the Km of the mutant PDD is about 3 times the Km of wild-type PDD for 1,3-propanediol. In another embodiment, the mutant PDD has a Km of about 80 mM for 1,3-propanediol. In a further embodiment, the mutant PDD is obtainable from  E.blattae  ATCC accession number PTA-92.  
           [0008]    In yet another embodiment of the present invention, the mutant PDD comprises a mutation corresponding to residue His105 to Leu in  E.blatte  PDD as shown in FIG. 3. In an additional embodiment, the mutant PDD comprises the amino acid shown in SEQ ID NO:2 and is encoded by nucleic acid having the sequence as shown in SEQ ID NO:1.  
           [0009]    The present invention also provides expression vectors and host cells comprising the isolated nucleic acid having the sequence as shown in SEQ ID NO:1. In one embodiment, the host cell includes Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces and Pseudomonas.  
           [0010]    In an additional aspect, the present invention relates to methods for producing 1,3-propanediol comprising the use of a microorganism comprising mutant PDD. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0011]    [0011]FIG. 1 provides the nucleic acid sequence (SEQ ID NO:1) for the mutant 1,3-propanediol dehydrogenase (PDD).  
         [0012]    [0012]FIG. 2 provides the amino acid sequence (SEQ ID NO:2) for the mutant 1,3-propanediol dehydrogenase (PDD).  
         [0013]    [0013]FIG. 3 provides an amino acid alignment of PDDs from various microorganisms. Eb_GEBT represents the ATCC deposited  E.blattae  mutant PDD, Eb — 429T and Eb — 907T are wild-type  E.blattae  (ATCC accession number 33429); Kpn is  Klebsiella pneumoniae  (GenBank accession # U30903); Cfu is  Citrobacter freundii  (GenBank accession number U09771) and Cpast is  Clostridium pasteurianum  (GenBank accession number AF006034).  
       DESCRIPTION OF THE MICROORGANISM DEPOSITS MADE UNDER THE BUDAPEST TREATY  
       [0014]    Applicants have made the following biological deposits under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for the Purposes of Patent Procedure:  
                                       Depositor Identification   International Depository           Reference   Designation   Date of Deposit                     Escherichia blattae     PTA-92   May 19, 1999       33429 derivative                  
 
       DETAILED DESCRIPTION  
     Definitions  
       [0015]    The terms “1,3-propanediol dehydrogenase” or “PDD” (also known in the art as “oxidoreductase”) refer to the polypeptide(s) responsible for an enzyme activity that is capable of catalyzing the reduction of 3-hydroxypropionaldehyde to 1,3-propanediol. 1,3-Propanediol dehydrogenase includes, for example, the polypeptide encoded by the dhaT gene. The present invention encompasses 3-propanediol dehydrogenase from any source including, but not limited to  E.blatte, K.pneumoniae, C.freundii, C.pasteurianum.    
         [0016]    As used herein, the term “mutant” or “mutation” refers to any genetic change that occurs in the nucleic acid of a microorganism and may or may not reflect a phenotypic change within the microorganism. A mutation may comprise a single base pair change, deletion or insertion; a mutation may comprise a change, deletion or insertion in a large number of base pairs; a mutation may also comprise a change in a large region of DNA, such as through duplication or inversion. The amino acid sequence of a mutant 1,3-propanediol dehydrogenase can be derived from a precursor 1,3-propanediol dehydrogenase by the substitution, deletion or insertion of one or more amino acids of the naturally occurring 1,3-propanediol dehydrogenase. Methods for modifying genes (e.g., through site-directed oligonucleotide mutagenesis) have been described in the art.  
         [0017]    The phrase “corresponding to” as used herein refers to the amino acid relatedness among 1,3-propanediol dehydrogenases as exemplified by FIG. 3. Specific residues discussed herein refer to an amino acid residue number which references the number assigned to the  E.blatte  GEB PDD shown in FIG. 3. The mutation of His to Leu is shown at residue 105 in FIG. 3. FIG. 3 illustrates that 1,3-propanediol dehydrogenases from a variety of microbial sources can be aligned using the algorithm CLUSTALW. The invention is not limited to the mutation of the  E.blattae  PDD shown in FIGS. 1 and 2, or the  E.blattae  deposited with the ATCC and having accession number PTA-92 but encompasses all PDDs containing amino acid residues at positions which are equivalent to the particular identified residue in  E.blattae.  A residue is equivalent if it is either homologous (i.e., corresponds in position for either the primary or tertiary structure) or analogous to a specific residue or portion of that residue in  E.blattae  PDD (i.e., having the same or similar functional capacity to combine, react, or interact chemically or structurally).  
         [0018]    In order to establish homology to primary structure, the amino acid sequence of a PDD is directly compared to the  E.blattae  PDD primary sequence (shown in FIG. 2) and particularly to a set of residues known to be invariant to all PDDs for which sequences are known (see, e.g., FIG. 3). The present invention encompasses the equivalent residue change in all sources of 1,3-propanediol dehydrogenase as long as the mutant form is able to alter the Km of the activity for 1,3-propanediol. In a preferred embodiment, the Km of the mutant form is increased for 1,3-propanediol. The nucleic acid sequence of SEQ ID NO:1 was obtained via PCR techniques. Such techniques are often characterized by inadvertent PCR generated sequence error. Therefore, the present invention also encompasses the 1,3-propanediol dehydrogenase of  E.blattae  having ATCC accession number PTA-92 and corresponding mutations in other microbial sources of the 1,3-propanediol dehydrogenases.  
         [0019]    The term “Km” refers to affinity of the enzyme for the substrate. A high Km reflects a low affinity; a low Km reflects a high affinity.  
         [0020]    The terms “carbon substrate” and “carbon source” refer to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.  
         [0021]    The terms “host cell” or “host organism” refer to a microorganism capable of receiving foreign or heterologous genes and of expressing those genes to produce an active gene product.  
         [0022]    As used herein, “nucleic acid” refers to a nucleotide or polynucleotide sequence, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be double-stranded or single-stranded, whether representing the sense or antisense strand. The terms “native” and “wild-type” refer to a gene as found in nature with its own regulatory sequences. As used herein “amino acid” refers to peptide or protein sequences or portions thereof.  
         [0023]    The term “expression” refers to the transcription and translation to gene product from a gene coding for the sequence of the gene product.  
         [0024]    The terms “plasmid”, “vector”, and “cassefte” refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.  
         [0025]    The terms “isolated” or “purified” as used herein refer to a nucleic acid or amino acid that is removed from at least one component with which it is naturally associated.  
       DETAILED DESCRIPTION OF THE INVENTION  
       [0026]    The present invention relates to mutant 1,3-propanediol dehydrogenase (PDD) characterized by having an increased Km for 1,3 propanediol.  
         [0027]    I. PDD Sequences  
         [0028]    Polynucleotide sequence as shown in SEQ ID NO:1 encodes the 1,3-propanediol dehydrogenase (SEQ ID NO:2) having the mutation of His to Leu at residue 105 as shown in FIG. 3. As will be understood by the skilled artisan, due to the degeneracy of the genetic code, a variety of polynucleotides can encode SEQ ID NO:2. The present invention encompasses all such polynucleotides. The present invention encompasses nucleic acid encoding PDD comprising a mutation corresponding to  E.blatte  residue His 105 to Leu as shown in FIG. 3. The nucleic acid and amino acid sequence for PDD from  K.pneumoniae  is given in GenBank accession number U30903; PDD from  C. freundii  is given in GenBank accession number U09771; for PDD from  C.pasteurianum  is given in GenBank accession number AF00034. The present invention also encompasses mutant PDD obtainable from  E.blattae  having ATCC accession number PTA-92.  
         [0029]    Methods of obtaining desired genes from a microbial genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA may be amplified using standard primer directed amplification methods such as polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for transformation using appropriate vectors.  
         [0030]    Alternatively, methods of using cosmid vectors for the transformation of suitable bacterial hosts are well described in Sambrook et al.,  Molecular Cloning: A Laboratory Manual,  Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbon, N.Y. (1989).  
         [0031]    Methods of making mutations in PDD genes are known to the skilled artisan and include for example site-directed mutagenesis, procedures described in United States patent U.S. Pat. No. 4,760,025 issued Jul. 26, 1988.  
         [0032]    Vectors and Expression Cassettes  
         [0033]    The present invention provides a variety of vectors and transformation and expression cassettes suitable for the cloning, transformation and expression of mutant PDD as well as other proteins associated with 1,3-propanediol production into a suitable host cell. Suitable vectors will be those which are compatible with the bacterium employed. Suitable vectors can be derived, for example, from a bacteria, a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, a yeast or a plant. Protocols for obtaining and using such vectors are known to those in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual—volumes 1,2,3 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)).  
         [0034]    Typically, the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.  
         [0035]    Initiation control regions or promoters, which are useful to drive expression of PDD in the desired host cell, are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, trp, IP L , IP R , T7, tac, and trc (useful for expression in  E. coli ).  
         [0036]    Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included.  
         [0037]    For effective expression of the instant enzymes, DNA encoding the enzymes are linked operably through initiation codons to selected expression control regions such that expression results in the formation of the appropriate messenger RNA.  
         [0038]    Transformation of Suitable Hosts and Expression of PDD  
         [0039]    Once suitable cassettes are constructed they are used to transform appropriate host cells. Introduction of the cassette containing mutant 1,3-propanediol dehydrogenase, either separately or together with other proteins necessary for the production of 1,3-propanediol, into the host cell may be accomplished by known procedures such as by transformation (e.g., using calcium-permeabilized cells, electroporation) or by transfection using a recombinant phage virus. (Sambrook et al., supra.).  
         [0040]    Host Cells  
         [0041]    Suitable host cells for the recombinant production of 1,3-propanediol may be either prokaryotic or eukaryotic and will be limited only by the host cell ability to express active enzymes. Preferred hosts will be those typically useful for production of glycerol or 1,3-propanediol such as Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces and Pseudomonas. Most preferred in the present invention are  E. coli,  Klebsiella species and Saccharomyces species.  
         [0042]    Media and Carbon Substrates  
         [0043]    Fermentation media in the present invention must contain suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose, or mixtures thereof, and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Additionally, the carbon substrate may also be one-carbon substrates such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated.  
         [0044]    Preferred carbon substrates are monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates. More preferred are sugars such as glucose, fructose, sucrose and single carbon substrates such as methanol and carbon dioxide. Most preferred is glucose.  
         [0045]    In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for glycerol production. Particular attention is given to Co(II) salts and/or vitamin B 12  or precursors thereof.  
         [0046]    Culture Conditions  
         [0047]    Typically, cells are grown at 30° C. in appropriate media. Preferred growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Malt Extract (YM) broth. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by someone skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2′:3′-monophosphate or cyclic adenosine 2′:5′-monophosphate, may also be incorporated into the reaction media. Similarly, the use of agents known to modulate enzymatic activities (e.g., sulphites, bisulphites and alkalis) that lead to enhancement of glycerol production may be used in conjunction with or as an alternative to genetic manipulations.  
         [0048]    Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as range for the initial condition.  
         [0049]    Reactions may be performed under aerobic or anaerobic conditions where anaerobic or microaerobic conditions are preferred.  
         [0050]    The manner and method of carrying out the present invention may be more fully understood by those of skill in the art by reference to the following examples, which examples are not intended in any manner to limit the scope of the present invention or of the claims directed thereto. 
     
    
     EXAMPLES  
       [0051]    Example 1 describes the kinetic changes associated with the mutant PDD shown in SEQ ID NO:2.  
         [0052]    Materials and Methods  
         [0053]    Strains—Wild type ATCC 33429,  E.blattae  comprising the mutant PDD ATCC accession number PTA-92.  
         [0054]    Growth—Cells were grown in a complex medium at 30C 500 ml in a 2800 ml fernbach with shaking at 225 rpm for 20 hr. The medium consists of KH2PO4, 5.4 g/L; (NH4)2SO4, 1.2 g/L; MgSO47H2O, 0.4 g/L; yeast extract, 2.0 g/L; tryptone, 2.0 g/L; and glycerol, 9.2 g/L in tap water. The pH was adjusted to 7.1 with KOH before autoclaving (Honda, et al., 1980, J. Bacteriol, 143:1458-1465).  
         [0055]    Extract prep—Cells were harvested by centrifugation with care to avoid anaerobic conditions. Pellets were resuspended in 100 mM Tricine pH 8.2 containing 50 mM KCl and 1 mM DTT. Cells were disrupted by passage through a French pressure cell. Crude extracts were clarified by centrifugation at 20K×g for 20 min followed by 100K×g for 1 hr to yield the high speed supernatant (HSS) fraction.  
         [0056]    Assays—the assay for PDD was performed as described by Johnson, E. A. et al., 1987, J. Bacteriol. 169:2050-2054.  
         [0057]    Partial purification of PDD—HSS was separated on a 16×100 Poros 20HQ column. The buffers were A, 50 mM HEPES, pH 7.4 containing 100 uM MnCl and B, A buffer containing 500 mM KCl. The column was loaded and developed at 10 ml/min. The gradient was 10 CV wash, a linear gradient to 70% B in 10 CV, and 1 CV to 100% B. The activity was detected in the very early fractions of the gradient. Pooled column fractions of the 33429 strain were used as collected for assays after the addition of additional of DTT to 1 mM. The active fractions from strain GEB031 were pooled and concentrated on a PM30 membrane and used as concentrated after the addition of additional 1 mM DTT.  
                                                           Strain   GD (U/mg)   PDD (U/mg)   Ratio GD/PDD                           33429   0.64   0.22   2.9           GEB031   0.79   0.08   9.9                      
 
         [0058]    PDD Kinetics—The results are shown below.  
                                                       Strain   Km (mM Propanediol)   Km (uM NAD)                           33429   28   57                      
 
         [0059]    Example 2: Cloning and sequencing the 1,3-propanediol dehydrogenase genes (dhaT) from  E. blattae.    
         [0060]    The dhaT genes were amplified by PCR from genomic DNA from  E. blattae  as template DNA using synthetic primers (primer 1 and primer 2) based on the  K. pneumoniae  dhaT sequence and incorporating an XbaI site at the 5′ end and a BamHI site at the 3′ end. The product was subcloned into pCR-Blunt II-TOPO (Invitrogen). The cloning dhaT were then sequenced was standard techniques.  
         [0061]    The results of the DNA sequencing are given in SEQ ID NO:1 and SEQ ID NO:2.  
                                   Primer 1           5′ TCTGATACGGGATCCTCAGAATGCCTGGCGGAAAAT3′                       Primer 2           5′ GCGCCGTCTAGAATTATGAGCTATCGTATGTTTGATTATCTG3′          
 
         [0062]    As will be readily understood by the skilled artisan, nucleic acid sequence generated via PCR methods may comprise inadvertent errors. The present invention also encompasses nucleic acid encoding PDD obtainable from  E.blattae  having ATCC accession number PTA-92.  
         [0063]    All references cited herein, including patents, patent applications, sequences and publications are hereby incorporated in their entirety by reference.  
     
       
       
         1 
         
           
             12  
           
           
             1  
             1164  
             DNA  
             Escherichia blattae  
           
            1 

atgagctatc gtatgtttga ttatctggtt ccaaatgtra acttctttgg cccgggcgcc     60 

gtttctgttg ttggccagcg ctgccagctg ctggggggta aaaaagccct gctggtgacc    120 

gataagggcc tgcgcgccat taaagacggt gctgtcgatc agaccgtgaa gcacctgaaa    180 

gccgccggta ttgaggtggt cattttcgac ggggtcgagc cgaacccgaa agacaccaac    240 

gtgctcgacg gcctggccat gttccgtaaa gagcagtgcg acatgataat caccgtcggc    300 

ggcggcagcc cgctcgactg cggtaaaggc attggtattg cggccaccca cccgggtgat    360 

ctgtacagct atgccggtat cgaaacactc accaacccgc tgccgcccat tattgcggtc    420 

aacaccaccg ccgggaccgc cagcgaagtc acccgccact gcgtgctgac taacaccaaa    480 

accaaagtaa aatttgtgat tgtcagctgg cgcaacctgc cttccgtctc cattaacgat    540 

ccgctgctga tgatcggcaa gcccgccggg ctgaccgccg ccaccggtat ggatgccctg    600 

acccacgcgg tagaggccta tatctccaaa gacgccaacc cggttaccga tgcctctgct    660 

attcaggcca tcaaactgat tgccaccaac ttgcgccagg ccgtcgccct ggggaccaac    720 

ctcaaagccc gtgaaaacat ggcctgcgcc tctctgctgg ccgggatggc ctttaacaac    780 

gccaacctgg gctatgttca cgccatggct caccagctgg gcggcctgta cgacatggcc    840 

cacggggtgg cgaacgcggt cctgctgccc catgtctgcc gctataacct gattgccaac    900 

ccggaaaaat ttgccgatat cgccaccttt atgggggaaa acaccaccgg tctttccacc    960 

atggacgcag cggagctggc catcagcgcc attgcccgtc tgtctaaaga tgtcgggatc   1020 

ccgcagcacc tgcgtgaact gggggtaaaa gaggccgact tcccgtacat ggcagaaatg   1080 

gccctgaaag acggcaacgc cttctctaac ccgcgcaaag ggaacgaaaa agagattgcc   1140 

gacattttcc gccaggcatt ctga                                          1164 

 
           
             2  
             387  
             PRT  
             Escherichia blattae  
           
            2 

Met Ser Tyr Arg Met Phe Asp Tyr Leu Val Pro Asn Val Asn Phe Phe 
 1               5                  10                  15 

Gly Pro Gly Ala Val Ser Val Val Gly Gln Arg Cys Gln Leu Leu Gly 
            20                  25                  30 

Gly Lys Lys Ala Leu Leu Val Thr Asp Lys Gly Leu Arg Ala Ile Lys 
        35                  40                  45 

Asp Gly Ala Val Asp Gln Thr Val Lys His Leu Lys Ala Ala Gly Ile 
    50                  55                  60 

Glu Val Val Ile Phe Asp Gly Val Glu Pro Asn Pro Lys Asp Thr Asn 
65                  70                  75                  80 

Val Leu Asp Gly Leu Ala Met Phe Arg Lys Glu Gln Cys Asp Met Ile 
                85                  90                  95 

Ile Thr Val Gly Gly Gly Ser Pro Leu Asp Cys Gly Lys Gly Ile Gly 
            100                 105                 110 

Ile Ala Ala Thr His Pro Gly Asp Leu Tyr Ser Tyr Ala Gly Ile Glu 
        115                 120                 125 

Thr Leu Thr Asn Pro Leu Pro Pro Ile Ile Ala Val Asn Thr Thr Ala 
    130                 135                 140 

Gly Thr Ala Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Lys 
145                 150                 155                 160 

Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val 
                165                 170                 175 

Ser Ile Asn Asp Pro Leu Leu Met Ile Gly Lys Pro Ala Gly Leu Thr 
            180                 185                 190 

Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu Ala Tyr Ile 
        195                 200                 205 

Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ser Ala Ile Gln Ala Ile 
    210                 215                 220 

Lys Leu Ile Ala Thr Asn Leu Arg Gln Ala Val Ala Leu Gly Thr Asn 
225                 230                 235                 240 

Leu Lys Ala Arg Glu Asn Met Ala Cys Ala Ser Leu Leu Ala Gly Met 
                245                 250                 255 

Ala Phe Asn Asn Ala Asn Leu Gly Tyr Val His Ala Met Ala His Gln 
            260                 265                 270 

Leu Gly Gly Leu Tyr Asp Met Ala His Gly Val Ala Asn Ala Val Leu 
        275                 280                 285 

Leu Pro His Val Cys Arg Tyr Asn Leu Ile Ala Asn Pro Glu Lys Phe 
    290                 295                 300 

Ala Asp Ile Ala Thr Phe Met Gly Glu Asn Thr Thr Gly Leu Ser Thr 
305                 310                 315                 320 

Met Asp Ala Ala Glu Leu Ala Ile Ser Ala Ile Ala Arg Leu Ser Lys 
                325                 330                 335 

Asp Val Gly Ile Pro Gln His Leu Arg Glu Leu Gly Val Lys Glu Ala 
            340                 345                 350 

Asp Phe Pro Tyr Met Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 
        355                 360                 365 

Ser Asn Pro Arg Lys Gly Asn Glu Lys Glu Ile Ala Asp Ile Phe Arg 
    370                 375                 380 

Gln Ala Phe 
385 

 
           
             3  
             36  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            3 

tctgatacgg gatcctcaga atgcctggcg gaaaat                               36 

 
           
             4  
             42  
             DNA  
             Artificial Sequence  
             
               primer  
             
           
            4 

gcgccgtcta gaattatgag ctatcgtatg tttgattatc tg                        42 

 
           
             5  
             1164  
             DNA  
             Escherichia blattae  
           
            5 

atgagctatc gtatgtttga ttatctggtt ccaaatgtga acttctttgg cccgggcgcc     60 

gtttctgttg ttggccagcg ctgccagctg ctggggggta aaaaagccct gctggtgacc    120 

gataagggcc tgcgcgccat taaagacggt gctgtcgatc agaccgtgaa gcacctgaaa    180 

gccgccggta ttgaggtggt cattttcgac ggggtcgagc cgaacccgaa agacaccaac    240 

gtgctcgacg gcctggccat gttccgtaaa gagcagtgcg acatgataat caccgtcggc    300 

ggcggcagcc cgcacgactg cggtaaaggc attggtattg cggccaccca cccgggtgat    360 

ctgtacagct atgccggtat cgaaacactc accaacccgc tgccgcccat tattgcggtc    420 

aacaccaccg ccgggaccgc cagcgaagtc acccgccact gcgtgctgac taacaccaaa    480 

accaaagtaa aatttgtgat tgtcagctgg cgcaacctgc cttccgtctc cattaacgat    540 

ccgctgctga tgatcggcaa gcccgccggg ctgaccgccg ccaccggtat ggatgccctg    600 

acccacgcgg tagaggccta tatctccaaa gacgccaacc cggttaccga tgcctctgct    660 

attcaggcca tcaaactgat tgccaccaac ttgcgccagg ccgtcgccct ggggaccaac    720 

ctcaaagccc gtgaaaacat ggcctgcgcc tctctgctgg ccgggatggc ctttaacaac    780 

gccaacctgg gctatgttca cgccatggct caccagctgg gcggcctgta cgacatggcc    840 

cacggggtgg cgaacgcggt cctgctgccc catgtctgcc gctataacct gattgccaac    900 

ccggaaaaat ttgccgatat cgccaccttt atgggggaaa acaccaccgg tctttccacc    960 

atggacgcag cggagctggc catcagcgcc attgcccgtc tgtctaaaga tgtcgggatc   1020 

ccgcagcacc tgcgtgaact gggggtaaaa gaggccgact tcccgtacat ggcagaaatg   1080 

gccctgaaag acggcaacgc cttctctaac ccgcgcaaag ggaacgaaaa agagattgcc   1140 

gacattttcc gccaggcatt ctga                                          1164 

 
           
             6  
             1164  
             DNA  
             Escherichia blattae  
           
            6 

atgagctatc gtatgtttga ttatctggtt ccaaatgtra acttctttgg cccgggcgcc     60 

gtttctgttg ttggccagcg ctgccagctg ctggggggta aaaaagccct gctggtgacc    120 

gataagggcc tgcgcgccat taaagacggt gctgtcgatc agaccgtgaa gcacctgaaa    180 

gccgccggta ttgaggtggt cattttcgac ggggtcgagc cgaacccgaa agacaccaac    240 

gtgctcgacg gcctggccat gttccgtaaa gagcagtgcg acatgataat caccgtcggc    300 

ggcggcagcc cgctcgactg cggtaaaggc attggtattg cggccaccca cccgggtgat    360 

ctgtacagct atgccggtat cgaaacactc accaacccgc tgccgcccat tattgcggtc    420 

aacaccaccg ccgggaccgc cagcgaagtc acccgccact gcgtgctgac taacaccaaa    480 

accaaagtaa aatttgtgat tgtcagctgg cgcaacctgc cttccgtctc cattaacgat    540 

ccgctgctga tgatcggcaa gcccgccggg ctgaccgccg ccaccggtat ggatgccctg    600 

acccacgcgg tagaggccta tatctccaaa gacgccaacc cggttaccga tgcctctgct    660 

attcaggcca tcaaactgat tgccaccaac ttgcgccagg ccgtcgccct ggggaccaac    720 

ctcaaagccc gtgaaaacat ggcctgcgcc tctctgctgg ccgggatggc ctttaacaac    780 

gccaacctgg gctatgttca cgccatggct caccagctgg gcggcctgta cgacatggcc    840 

cacggggtgg cgaacgcggt cctgctgccc catgtctgcc gctataacct gattgccaac    900 

ccggaaaaat ttgccgatat cgccaccttt atgggggaaa acaccaccgg tctttccacc    960 

atggacgcag cggagctggc catcagcgcc attgcccgtc tgtctaaaga tgtcgggatc   1020 

ccgcagcacc tgcgtgaact gggggtaaaa gaggccgact tcccgtacat ggcagaaatg   1080 

gccctgaaag acggcaacgc cttctctaac ccgcgcaaag ggaacgaaaa agagattgcc   1140 

gacattttcc gccaggcatt ctga                                          1164 

 
           
             7  
             387  
             PRT  
             Escherichia blattae  
           
            7 

Met Ser Tyr Arg Met Phe Asp Tyr Leu Val Pro Asn Val Asn Phe Phe 
 1               5                  10                  15 

Gly Pro Gly Ala Val Ser Val Val Gly Gln Arg Cys Gln Leu Leu Gly 
            20                  25                  30 

Gly Lys Lys Ala Leu Leu Val Thr Asp Lys Gly Leu Arg Ala Ile Lys 
        35                  40                  45 

Asp Gly Ala Val Asp Gln Thr Val Lys His Leu Lys Ala Ala Gly Ile 
    50                  55                  60 

Glu Val Val Ile Phe Asp Gly Val Glu Pro Asn Pro Lys Asp Thr Asn 
65                  70                  75                  80 

Val Leu Asp Gly Leu Ala Met Phe Arg Lys Glu Gln Cys Asp Met Ile 
                85                  90                  95 

Ile Thr Val Gly Gly Gly Ser Pro His Asp Cys Gly Lys Gly Ile Gly 
            100                 105                 110 

Ile Ala Ala Thr His Pro Gly Asp Leu Tyr Ser Tyr Ala Gly Ile Glu 
        115                 120                 125 

Thr Leu Thr Asn Pro Leu Pro Pro Ile Ile Ala Val Asn Thr Thr Ala 
    130                 135                 140 

Gly Thr Ala Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Lys 
145                 150                 155                 160 

Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val 
                165                 170                 175 

Ser Ile Asn Asp Pro Leu Leu Met Ile Gly Lys Pro Ala Gly Leu Thr 
            180                 185                 190 

Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu Ala Tyr Ile 
        195                 200                 205 

Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ser Ala Ile Gln Ala Ile 
    210                 215                 220 

Lys Leu Ile Ala Thr Asn Leu Arg Gln Ala Val Ala Leu Gly Thr Asn 
225                 230                 235                 240 

Leu Lys Ala Arg Glu Asn Met Ala Cys Ala Ser Leu Leu Ala Gly Met 
                245                 250                 255 

Ala Phe Asn Asn Ala Asn Leu Gly Tyr Val His Ala Met Ala His Gln 
            260                 265                 270 

Leu Gly Gly Leu Tyr Asp Met Ala His Gly Val Ala Asn Ala Val Leu 
        275                 280                 285 

Leu Pro His Val Cys Arg Tyr Asn Leu Ile Ala Asn Pro Glu Lys Phe 
    290                 295                 300 

Ala Asp Ile Ala Thr Phe Met Gly Glu Asn Thr Thr Gly Leu Ser Thr 
305                 310                 315                 320 

Met Asp Ala Ala Glu Leu Ala Ile Ser Ala Ile Ala Arg Leu Ser Lys 
                325                 330                 335 

Asp Val Gly Ile Pro Gln His Leu Arg Glu Leu Gly Val Lys Glu Ala 
            340                 345                 350 

Asp Phe Pro Tyr Met Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 
        355                 360                 365 

Ser Asn Pro Arg Lys Gly Asn Glu Lys Glu Ile Ala Asp Ile Phe Arg 
    370                 375                 380 

Gln Ala Phe 
385 

 
           
             8  
             387  
             PRT  
             Escherichia blattae  
           
            8 

Met Ser Tyr Arg Met Phe Asp Tyr Leu Val Pro Asn Val Asn Phe Phe 
 1               5                  10                  15 

Gly Pro Gly Ala Val Ser Val Val Gly Gln Arg Cys Gln Leu Leu Gly 
            20                  25                  30 

Gly Lys Lys Ala Leu Leu Val Thr Asp Lys Gly Leu Arg Ala Ile Lys 
        35                  40                  45 

Asp Gly Ala Val Asp Gln Thr Val Lys His Leu Lys Ala Ala Gly Ile 
    50                  55                  60 

Glu Val Val Ile Phe Asp Gly Val Glu Pro Asn Pro Lys Asp Thr Asn 
65                  70                  75                  80 

Val Leu Asp Gly Leu Ala Met Phe Arg Lys Glu Gln Cys Asp Met Ile 
                85                  90                  95 

Ile Thr Val Gly Gly Gly Ser Pro Leu Asp Cys Gly Lys Gly Ile Gly 
            100                 105                 110 

Ile Ala Ala Thr His Pro Gly Asp Leu Tyr Ser Tyr Ala Gly Ile Glu 
        115                 120                 125 

Thr Leu Thr Asn Pro Leu Pro Pro Ile Ile Ala Val Asn Thr Thr Ala 
    130                 135                 140 

Gly Thr Ala Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Lys 
145                 150                 155                 160 

Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val 
                165                 170                 175 

Ser Ile Asn Asp Pro Leu Leu Met Ile Gly Lys Pro Ala Gly Leu Thr 
            180                 185                 190 

Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu Ala Tyr Ile 
        195                 200                 205 

Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ser Ala Ile Gln Ala Ile 
    210                 215                 220 

Lys Leu Ile Ala Thr Asn Leu Arg Gln Ala Val Ala Leu Gly Thr Asn 
225                 230                 235                 240 

Leu Lys Ala Arg Glu Asn Met Ala Cys Ala Ser Leu Leu Ala Gly Met 
                245                 250                 255 

Ala Phe Asn Asn Ala Asn Leu Gly Tyr Val His Ala Met Ala His Gln 
            260                 265                 270 

Leu Gly Gly Leu Tyr Asp Met Ala His Gly Val Ala Asn Ala Val Leu 
        275                 280                 285 

Leu Pro His Val Cys Arg Tyr Asn Leu Ile Ala Asn Pro Glu Lys Phe 
    290                 295                 300 

Ala Asp Ile Ala Thr Phe Met Gly Glu Asn Thr Thr Gly Leu Ser Thr 
305                 310                 315                 320 

Met Asp Ala Ala Glu Leu Ala Ile Ser Ala Ile Ala Arg Leu Ser Lys 
                325                 330                 335 

Asp Val Gly Ile Pro Gln His Leu Arg Glu Leu Gly Val Lys Glu Ala 
            340                 345                 350 

Asp Phe Pro Tyr Met Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 
        355                 360                 365 

Ser Asn Pro Arg Lys Gly Asn Glu Lys Glu Ile Ala Asp Ile Phe Arg 
    370                 375                 380 

Gln Ala Phe 
385 

 
           
             9  
             387  
             PRT  
             Escherichia blattae  
           
            9 

Met Ser Tyr Arg Met Phe Asp Tyr Leu Val Pro Asn Val Asn Phe Phe 
 1               5                  10                  15 

Gly Pro Gly Ala Val Ser Val Val Gly Gln Arg Cys Gln Leu Leu Gly 
            20                  25                  30 

Gly Lys Lys Ala Leu Leu Val Thr Asp Lys Gly Leu Arg Ala Ile Lys 
        35                  40                  45 

Asp Gly Ala Val Asp Gln Thr Val Lys His Leu Lys Ala Ala Gly Ile 
    50                  55                  60 

Glu Val Val Ile Phe Asp Gly Val Glu Pro Asn Pro Lys Asp Thr Asn 
65                  70                  75                  80 

Val Leu Asp Gly Leu Ala Met Phe Arg Lys Glu Gln Cys Asp Met Ile 
                85                  90                  95 

Ile Thr Val Gly Gly Gly Ser Pro His Asp Cys Gly Lys Gly Ile Gly 
            100                 105                 110 

Ile Ala Ala Thr His Pro Gly Asp Leu Tyr Ser Tyr Ala Gly Ile Glu 
        115                 120                 125 

Thr Leu Thr Asn Pro Leu Pro Pro Ile Ile Ala Val Asn Thr Thr Ala 
    130                 135                 140 

Gly Thr Ala Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Lys 
145                 150                 155                 160 

Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val 
                165                 170                 175 

Ser Ile Asn Asp Pro Leu Leu Met Ile Ser Lys Pro Ala Gly Leu Thr 
            180                 185                 190 

Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu Ala Tyr Ile 
        195                 200                 205 

Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ser Ala Ile Gln Ala Ile 
    210                 215                 220 

Lys Leu Ile Ala Thr Asn Leu Arg Gln Ala Val Ala Leu Gly Thr Asn 
225                 230                 235                 240 

Leu Lys Ala Arg Glu Asn Met Ala Cys Ala Ser Leu Leu Ala Gly Met 
                245                 250                 255 

Ala Phe Asn Asn Ala Asn Leu Gly Tyr Val His Ala Met Ala His Gln 
            260                 265                 270 

Leu Gly Gly Leu Tyr Asp Met Ala His Gly Val Ala Asn Ala Val Leu 
        275                 280                 285 

Leu Pro His Val Cys Arg Tyr Asn Leu Ile Ala Asn Pro Glu Lys Phe 
    290                 295                 300 

Ala Asp Ile Ala Thr Phe Met Gly Glu Asn Thr Thr Gly Leu Ser Thr 
305                 310                 315                 320 

Met Asp Ala Ala Glu Leu Ala Ile Ser Ala Ile Ala Arg Leu Ser Lys 
                325                 330                 335 

Asp Val Gly Ile Pro Gln His Leu Arg Glu Leu Gly Val Lys Glu Ala 
            340                 345                 350 

Asp Phe Pro Tyr Met Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 
        355                 360                 365 

Ser Asn Pro Arg Lys Gly Asn Glu Lys Glu Ile Ala Asp Ile Phe Arg 
    370                 375                 380 

Gln Ala Phe 
385 

 
           
             10  
             387  
             PRT  
             Klebsiella pneumoniae  
           
            10 

Met Ser Tyr Arg Met Phe Asp Tyr Leu Val Pro Asn Val Asn Phe Phe 
 1               5                  10                  15 

Gly Pro Asn Ala Ile Ser Val Val Gly Glu Arg Cys Gln Leu Leu Gly 
            20                  25                  30 

Gly Lys Lys Ala Leu Leu Val Thr Asp Lys Gly Leu Arg Ala Ile Lys 
        35                  40                  45 

Asp Gly Ala Val Asp Lys Thr Leu His Tyr Leu Arg Glu Ala Gly Ile 
    50                  55                  60 

Glu Val Ala Ile Phe Asp Gly Val Glu Pro Asn Pro Lys Asp Thr Asn 
65                  70                  75                  80 

Val Arg Asp Gly Leu Ala Val Phe Arg Arg Glu Gln Cys Asp Ile Ile 
                85                  90                  95 

Val Thr Val Gly Gly Gly Ser Pro His Asp Cys Gly Lys Gly Ile Gly 
            100                 105                 110 

Ile Ala Ala Thr His Glu Gly Asp Leu Tyr Gln Tyr Ala Gly Ile Glu 
        115                 120                 125 

Thr Leu Thr Asn Pro Leu Pro Pro Ile Val Ala Val Asn Thr Thr Ala 
    130                 135                 140 

Gly Thr Ala Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Glu 
145                 150                 155                 160 

Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val 
                165                 170                 175 

Ser Ile Asn Asp Pro Leu Leu Met Ile Gly Lys Pro Ala Ala Leu Thr 
            180                 185                 190 

Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu Ala Tyr Ile 
        195                 200                 205 

Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ala Ala Met Gln Ala Ile 
    210                 215                 220 

Arg Leu Ile Ala Arg Asn Leu Arg Gln Ala Val Ala Leu Gly Ser Asn 
225                 230                 235                 240 

Leu Gln Ala Arg Glu Asn Met Ala Tyr Ala Ser Leu Leu Ala Gly Met 
                245                 250                 255 

Ala Phe Asn Asn Ala Asn Leu Gly Tyr Val His Ala Met Ala His Gln 
            260                 265                 270 

Leu Gly Gly Leu Tyr Asp Met Pro His Gly Val Ala Asn Ala Val Leu 
        275                 280                 285 

Leu Pro His Val Ala Arg Tyr Asn Leu Ile Ala Asn Pro Glu Lys Phe 
    290                 295                 300 

Ala Asp Ile Ala Glu Leu Met Gly Glu Asn Ile Thr Gly Leu Ser Thr 
305                 310                 315                 320 

Leu Asp Ala Ala Glu Lys Ala Ile Ala Ala Ile Thr Arg Leu Ser Met 
                325                 330                 335 

Asp Ile Gly Ile Pro Gln His Leu Arg Asp Leu Gly Val Lys Glu Ala 
            340                 345                 350 

Asp Phe Pro Tyr Met Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 
        355                 360                 365 

Ser Asn Pro Arg Lys Gly Asn Glu Gln Glu Ile Ala Ala Ile Phe Arg 
    370                 375                 380 

Gln Ala Phe 
385 

 
           
             11  
             387  
             PRT  
             Citrobacter freundii  
           
            11 

Met Ser Tyr Arg Met Phe Asp Tyr Leu Val Pro Asn Val Asn Phe Phe 
 1               5                  10                  15 

Gly Pro Asn Ala Ile Ser Val Val Gly Glu Arg Cys Lys Leu Leu Gly 
            20                  25                  30 

Gly Lys Lys Ala Leu Leu Val Thr Asp Lys Gly Leu Arg Ala Ile Lys 
        35                  40                  45 

Asp Gly Ala Val Asp Lys Thr Leu Thr His Leu Arg Glu Ala Gly Ile 
    50                  55                  60 

Asp Val Val Val Phe Asp Gly Val Glu Pro Asn Pro Lys Asp Thr Asn 
65                  70                  75                  80 

Val Arg Asp Gly Leu Glu Val Phe Arg Lys Glu His Cys Asp Ile Ile 
                85                  90                  95 

Val Thr Val Gly Gly Gly Ser Pro His Asp Cys Gly Lys Gly Ile Gly 
            100                 105                 110 

Ile Ala Ala Thr His Glu Gly Asp Leu Tyr Ser Tyr Ala Gly Ile Glu 
        115                 120                 125 

Thr Leu Thr Asn Pro Leu Pro Pro Ile Val Ala Val Asn Thr Thr Ala 
    130                 135                 140 

Gly Thr Ala Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Lys 
145                 150                 155                 160 

Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val 
                165                 170                 175 

Ser Ile Asn Asp Pro Leu Leu Met Leu Gly Lys Pro Ala Pro Leu Thr 
            180                 185                 190 

Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu Ala Tyr Ile 
        195                 200                 205 

Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ala Ala Ile Gln Ala Ile 
    210                 215                 220 

Arg Leu Ile Ala Arg Asn Leu Arg Gln Ala Val Ala Leu Gly Ser Asn 
225                 230                 235                 240 

Leu Lys Ala Arg Glu Asn Met Ala Tyr Ala Ser Leu Leu Ala Gly Met 
                245                 250                 255 

Ala Phe Asn Asn Ala Asn Leu Gly Tyr Val His Ala Met Ala His Gln 
            260                 265                 270 

Leu Gly Gly Leu Tyr Asp Met Pro His Gly Val Ala Asn Ala Val Leu 
        275                 280                 285 

Leu Pro His Val Ala Arg Tyr Asn Leu Ile Ala Asn Pro Glu Lys Phe 
    290                 295                 300 

Ala Asp Ile Ala Glu Phe Met Gly Glu Asn Thr Asp Gly Leu Ser Thr 
305                 310                 315                 320 

Met Asp Ala Ala Glu Leu Ala Ile His Ala Ile Ala Arg Leu Ser Ala 
                325                 330                 335 

Asp Ile Gly Ile Pro Gln His Leu Arg Asp Leu Gly Val Lys Glu Ala 
            340                 345                 350 

Asp Phe Pro Tyr Met Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 
        355                 360                 365 

Ser Asn Pro Arg Lys Gly Asn Glu Lys Glu Ile Ala Glu Ile Phe Arg 
    370                 375                 380 

Gln Ala Phe 
385 

 
           
             12  
             385  
             PRT  
             Clostridium pasteurianum  
           
            12 

Met Arg Met Tyr Asp Phe Leu Ala Pro Asn Val Asn Phe Met Gly Ala 
 1               5                  10                  15 

Gly Ala Ile Lys Leu Val Gly Glu Arg Cys Lys Ile Leu Gly Gly Lys 
            20                  25                  30 

Lys Ala Leu Ile Val Thr Asp Lys Phe Leu Arg Asn Met Glu Asp Gly 
        35                  40                  45 

Ala Val Ala Gln Thr Val Lys Tyr Ile Lys Glu Ala Gly Ile Asp Val 
    50                  55                  60 

Ala Phe Tyr Asp Asp Val Glu Pro Asn Pro Lys Asp Thr Asn Val Arg 
65                  70                  75                  80 

Asp Gly Leu Lys Val Tyr Arg Lys Glu Asn Cys Asp Leu Ile Val Thr 
                85                  90                  95 

Val Gly Gly Gly Ser Ser His Asp Cys Gly Lys Gly Ile Gly Ile Ala 
            100                 105                 110 

Ala Thr His Glu Gly Asp Leu Tyr Asp Tyr Ala Gly Ile Glu Thr Leu 
        115                 120                 125 

Thr Asn Pro Leu Pro Pro Ile Val Ala Val Asn Thr Thr Ala Gly Thr 
    130                 135                 140 

Gly Ser Glu Val Thr Arg His Cys Val Ile Thr Asn Thr Lys Thr Lys 
145                 150                 155                 160 

Ile Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Leu Val Ser Ile 
                165                 170                 175 

Asn Asp Pro Ile Leu Met Ile Lys Lys Pro Ala Gly Leu Thr Ala Ala 
            180                 185                 190 

Thr Gly Met Asp Ala Leu Thr His Ala Ile Glu Ser Tyr Val Ser Lys 
        195                 200                 205 

Asp Ala Asn Pro Val Thr Asp Ala Leu Ala Ile Gln Ala Ile Lys Leu 
    210                 215                 220 

Ile Ala Asn Asn Leu Arg Gln Ala Val Ala Leu Gly Glu Asn Leu Glu 
225                 230                 235                 240 

Ala Arg Glu Asn Met Ala Tyr Ala Ser Leu Leu Ala Gly Met Ala Phe 
                245                 250                 255 

Asn Asn Ala Asn Leu Gly Tyr Val His Ala Met Ala His Gln Leu Gly 
            260                 265                 270 

Gly Leu Tyr Asp Met Ala His Gly Val Ala Asn Ala Met Leu Leu Pro 
        275                 280                 285 

His Val Glu Arg Tyr Asn Leu Ile Ser Asn Pro Lys Lys Phe Ala Asp 
    290                 295                 300 

Ile Ala Glu Phe Met Gly Glu Asn Ile Glu Gly Leu Ser Val Met Glu 
305                 310                 315                 320 

Ala Ala Glu Lys Ala Ile Asp Ala Met Phe Arg Leu Ser Lys Asp Val 
                325                 330                 335 

Gly Ile Pro Ala Ser Leu Lys Glu Met Gly Val Asn Glu Gly Asp Phe 
            340                 345                 350 

Glu Tyr Met Ala Lys Met Ala Leu Lys Asp Gly Asn Ala Phe Ser Asn 
        355                 360                 365 

Pro Arg Lys Gly Asn Glu Lys Asp Ile Val Lys Ile Phe Arg Glu Ala 
    370                 375                 380 

Phe 
385