The resonant recognition model (RRM) predicts amino acid residues in highly conserved regions of the hormone prolactin (PRL)

The resonant recognition model (RRM) is a model which treats the protein sequence as a discrete signal. It has been shown previously that certain periodicities (frequencies) in this signal characterise protein biological function. The RRM was employed to determine the characteristic frequencies of the hormone prolactin (PRL), and to identify amino acids ('hot spots') mostly contributing to these frequencies and thus proposed to mostly contribute to the biological function. The predicted 'hot spot' amino acids, Phe-19, Ser-26, Ser-33, Phe-37, Phe-40, Gly-47, Gly-49, Phe-50, Ser-61, Gly-129, Arg-176, Arg-177, Cys-191 and Arg-192 are found in the highly conserved amino-terminal and C-terminus regions of PRL. Our predictions agree with previous experimentally tested residues by site-direct mutagenesis and photoaffinity labelling.     

Analysis of beta-tubulin sequences reveals highly conserved, coordinated amino acid substitutions. Evidence that these 'hot spots' are directly involved in the conformational change required for dynamic instability

Vertebrate beta-tubulins have been classified into six classes on the basis of their C-terminal sequences [(1987) J. Cell Biol. 105, 1707-1720]. In particular, the sequences starting at residue 430 differ between isotypes of the same animal but are conserved between species. We extend this analysis and show that there are three 'hot spots', at residues 35, 55-57 and 124 which exhibit intra-species heterogeneity but inter-species conservation. There is a remarkable correlation between the identity of these residues and the C-terminal sequences, and suggests that the vertebrate beta-tubulins fall into three broad types. This correlation extends to those non-vertebrate organisms which have the Type 1 C-terminal sequence. We propose that these three 'hot spots' and the C-terminal peptide interact in the tertiary structure. We have also noted that the C-terminal peptide almost always contains a single phenylalanine or tyrosine residue, and that there is a strong correlation between this residue and the amino acids at positions 217/218, in both the vertebrate and non-vertebrate sequences. We propose that the C-terminal aromatic amino acid interacts with residues 217/218 in the tertiary structure. Analysis of conditions which stabilise microtubules and/or lower the steady state critical concentration strongly suggests that these two sets of coordinated amino acid substitutions are directly involved in effecting the conformational change associated with GTP hydrolysis which results in dynamic instability. We propose that there is an interaction between the highly acidic sequence between residue 430 and the aromatic amino acid (termed peptide A) and conserved basic amino acids located close to the 'hot spots'. We suggest that this interaction is altered in response to the assembly-dependent GTP hydrolysis, with the consequential increase in the subunit dissociation rate constant.     

Prediction of 'hot spots' in SV40 enhancer and relation with experimental data

A theoretical method for prediction of the points in the sequence which are relevant for its biological function, the so-called 'hot spots', is presented. This method is based on significant correlation between the spectrum of numerical presentation of any genetic sequence and its biological function. One number corresponds to the particular nucleotide, thus forming a numerical sequence. The 'hot spot' prediction has been tested on the SV40 enhancer as a model system. The SV40 enhancer was chosen because of existing detailed data about activities of systematically obtained mutants. These results have been compared with results obtained theoretically.     

Identification of residues in a hydrophilic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen.

The self-incompatibility response involves S allele-specific recognition between stigmatic S proteins and incompatible pollen. This response results in pollen inhibition. Defining the amino acid residues within the stigmatic S proteins that participate in S allele-specific inhibition of incompatible pollen is essential for the elucidation of the molecular basis of the self-incompatibility response. We have constructed mutant derivatives of the S1 protein from Papaver rhoeas by using site-directed mutagenesis and have tested their biological activity. This has enabled us to identify amino acid residues in the stigmatic S proteins of P. rhoeas that are required for S-specific inhibition of incompatible pollen. We report here the identification of several amino acid residues in the predicted hydrophilic loop 6 of the P. rhoeas stigmatic S1 protein that are involved in the inhibition of S1 pollen. Mutation of the only hypervariable amino acid, which is situated in this loop, resulted in the complete loss of ability of the S protein to inhibit S1 pollen. This clearly demonstrates that this residue plays a crucial role in pollen recognition and may also participate in defining allelic specificity. We have also established the importance of highly conserved amino acids adjacent to this hypervariable site. Our studies demonstrate that both variable and conserved amino acids in the region of the S protein corresponding to surface loop 6 are key elements that play a role in the recognition and inhibition of incompatible pollen in the pollen-pistil self-incompatibility reaction.     

The isolation and characterization of rabbit motilin precursor cDNA.

The cDNA sequence of rabbit motilin precursor has been determined. The predicted amino acid sequence indicates that the precursor consists of 133 amino acids and includes a 25 amino acid signal peptide followed by the 22 amino acid motilin sequence and an 86 amino acid motilin associated peptide (MAP). As in the human and porcine precursors, two lysine residues follow motilin in the rabbit sequence. Rabbit motilin shares 64% amino acid sequence identity with human and porcine motilin, and all amino acid substitutions represent conservative changes. Amino acid sequence alignments of the rabbit, human and porcine MAP sequences suggest three functional/structural motifs corresponding to a putative endoproteinase recognition site, a putative PEST site and a potential posttranslational processing recognition element.     

A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides

Presecretory signal peptides of 39 proteins from diverse prokaryotic and eukaryotic sources have been compared. Although varying in length and amino acid composition, the labile peptides share a hydrophobic core of approximately 12 amino acids. A positively charged residue (Lys or Arg) usually precedes the hydrophobic core. Core termination is defined by the occurrence of a charged residue, a sequence of residues which may induce a beta-turn in a polypeptide, or an interruption in potential alpha-helix or beta-extended strand structure. The hydrophobic cores contain, by weight average, 37% Leu: 15% Ala: 10% Val: 10% Phe: 7% Ile plus 21% other hydrophobic amino acids arranged in a non-random sequence. Following the hydrophobic cores (aligned by their last residue) a highly non-random and localized distribution of Ala is apparent within the initial eight positions following the core: (formula; see text) Coincident with this observation, Ala-X-Ala is the most frequent sequence preceding signal peptidase cleavage. We propose the existence of a signal peptidase recognition sequence A-X-B with the preferred cleavage site located after the sixth amino acid following the core sequence. Twenty-two of the above 27 underlined Ala residues would participate as A or B in peptidase cleavage. Position A includes the larger aliphatic amino acids, Leu, Val and Ile, as well as the residues already found at B (principally Ala, Gly and Ser). Since a preferred cleavage site can be discerned from carboxyl and not amino terminal alignment of the hydrophobic cores it is proposed that the carboxyl ends are oriented inward toward the lumen of the endoplasmic reticulum where cleavage is thought to occur. This orientation coupled with the predicted beta-turn typically found between the core and the cleavage site implies reverse hairpin insertion of the signal sequence. The structural features which we describe should help identify signal peptides and cleavage sites in presumptive amino acid sequences derived from DNA sequences.     

Probing the structure of the PI-SceI-DNA complex by affinity cleavage and affinity photocross-linking

The PI-SceI protein is an intein-encoded homing endonuclease that initiates the mobility of its gene by making a double strand break at a single site in the yeast genome. The PI-SceI protein splicing and endonucleolytic active sites are separately located in each of two domains in the PI-SceI structure. To determine the spatial relationship between bases in the PI-SceI recognition sequence and selected PI-SceI amino acids, the PI-SceI-DNA complex was probed by photocross-linking and affinity cleavage methods. Unique solvent-accessible cysteine residues were introduced into the two PI-SceI domains at positions 91, 97, 170, 230, 376, and 378, and the mutant proteins were modified with either 4-azidophenacyl bromide or iron (S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE). The phenyl azide-coupled proteins cross-linked to the PI-SceI target sequence, and the FeBABE-modified proteins cleaved the DNA proximal to the derivatized amino acid. The results suggest that an extended beta-hairpin loop in the endonuclease domain that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site. Conversely, residues 91, 97, and 170 in the protein splicing domain are in close proximity to a distant region of the substrate. To interpret our results, we used a new PI-SceI structure that is ordered in regions of the protein that bind DNA. The data strongly support a model of the PI-SceI-DNA complex derived from this structure.     

Complete amino acid sequence of rat L-type pyruvate kinase deduced from the cDNA sequence

cDNA clones, containing the entire coding region of rat L-type pyruvate kinase, were isolated and their nucleotide sequences were determined by the dideoxy-chain-termination method. The predicted coding region, which spans 543 amino acids, established the complete amino acid sequence of the L-type isozyme of pyruvate kinase for the first time. The deduced amino acid sequence of the L type has one phosphorylation site in its amino terminus and shows about 68% and 48% homologies with M1-type pyruvate kinase of chicken and yeast pyruvate kinase respectively. Domain A exhibits higher homology than domains B and C. The residues in the active site of the L-type enzyme of rats, lying between domains B and A2, are rather different from those of the M1-type enzyme of chickens, but other residues constituting the active site are identical with those of the chicken M1 type except for one amino acid substitution.     

α-Amylase structure and activity

Two computerized methods of predicting protein secondary structure from amino acid sequences are evaluated by using them on the α-amylase ofAspergillus oryzae, for which the three-dimensional structure has been determined. The methods are then used, with amino acid alignments, to predict the structures of other α-amylases. It is found that all α-amylases of known amino acid sequence have the same basic structure, a barrel of eight parallel stretches of extended chain surrounded by eight helices. Strong similarities are found in those areas of the proteins believed to bind an essential calcium ion and at that part of the active site that catalyzes bond hydrolysis in the substrates. The active site, as a whole, is formed mainly of amino acids situated on loops joining extended chain to the adjacent helix. Variations in the length and amino acid sequence of these loops, from one α-amylase to another, provide the differences in binding the substrates believed to account for the known variations in action pattern of α-amylases of different biological origins.     

Thermolabile alkaline phosphatase from Northern shrimp (Pandalus borealis): protein and cDNA sequence analyses

Sequence analysis of short fragments resulting from trypsin digestion of the thermolabile shrimp alkaline phosphatase (SAP) from Northern shrimp Pandalus borealis formed the basis for amplification of its encoding cDNA. The predicted protein sequence was recognized as containing the consensus alkaline phosphatase motif comprising the active site of this protein family. Protein sequence homology searches identified several eukaryote alkaline phosphatases with which the 475-amino acid SAP polypeptide revealed shares 45% amino acid sequence identity. Residues for potential metal binding seem to be conserved in these proteins. The predicted 54-kDa molecular mass of SAP is smaller than previously reported, but is consistent with our recent SDS-PAGE analysis of the native protein. Compared to its homologs, the shrimp enzyme has a surplus of negatively charged amino acids, while the relative number of prolines is lower and the frequency of aromatic residues is higher than in mesophilic counterparts.     

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

Reading the three-dimensional structure of lattice model-designed proteins from their amino acid sequence.

While all the information required for the folding of a protein is contained in its amino acid sequence, one has not yet learned how to extract this information to predict the detailed, biological active, three-dimensional structure of a protein whose sequence is known. Using insight obtained from lattice model simulations of the folding of small proteins (fewer than 100 residues), in particular of the fact that this phenomenon is essentially controlled by conserved contacts (Mirny et al., Proc Natl Acad Sci USA 1995;92:1282) among (few) strongly interacting ("hot") amino acids (Tiana et al., J Chem Phys 1998;108:757-761), which also stabilize local elementary structures formed early in the folding process and leading to the (postcritical) folding core when they assemble together (Broglia et al., Proc Natl Acad Sci USA 1998;95:12930, Broglia & Tiana, J Chem Phys 2001;114:7267), we have worked out a successful strategy for reading the three-dimensional structure of lattice model-designed proteins from the knowledge of only their amino acid sequence and of the contact energies among the amino acids.     

Structural and functional studies of the amino terminus of yeast metallothionein

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.     

Molecular cloning and expression of cDNA for rat pancreatic cholesterol esterase

A full-length cDNA complementary to the rat pancreatic cholesterol esterase mRNA was isolated by screening a rat pancreatic cDNA expression library in lambda gt11 vector with antibodies against the porcine pancreatic cholesterol esterase. The isolated cholesterol esterase cDNA is 2050 bp in length and contains an open reading frame coding for a protein of 612 amino acids. A 20-amino acid hydrophobic leader sequence is predicted, based on the position of the first ATG initiation codon upstream from the sequenced amino terminus of the isolated cholesterol esterase. The cholesterol esterase cDNA was subcloned into a mammalian expression vector, pSVL, for transfection studies. Expression of the cDNA in COS cells resulted in the production of bile salt-stimulated cholesterol esterase. Comparison of the cholesterol esterase cDNA sequence with other proteins revealed that the pancreatic cholesterol esterase is identical to rat pancreatic lysophospholipase. The primary structure of cholesterol esterase displayed no significant homology with other lipases, although the putative lipid interfacial recognition site of G-X-S-X-G is present in the cholesterol esterase sequence. However, the cholesterol esterase sequence revealed a 63-amino-acid domain which is highly homologous to the active site domain of other serine esterases. These data suggest that cholesterol esterase may be a member of the serine esterase supergene family. Analysis of the cholesterol esterase structure also revealed a repetitive sequence enriched with Pro, Asp, Glu, Ser, and Thr residues at the C-terminal end of the protein. This sequence is reminiscent of the PEST-rich sequences in short-lived proteins, suggesting that cholesterol esterase may have a short half-life in vivo. Northern blot hybridization showed that the bile salt-stimulated cholesterol esterase mRNA is present in liver suggesting that this protein may also be synthesized by liver cells.     

Engineering the C-terminus of firefly luciferase as an indicator of covalent modification of proteins

Protein kinase recognition sequences and proteinase sites were engineered into the cDNA encoding firefly luciferase from Photinus pyralis in order to establish whether these modified proteins could be developed as bioluminescent indicators of covalent modification of proteins. Two key domains of the luciferase were modified in order to identify regions of the protein in which peptide sequences may be engineered whilst retaining bioluminescent activity; one between amino acids 209 and 227 and the other at the C-terminus, between amino acids 537 and 550. Mutation of amino acids between residues 209 and 227 reduced bioluminescent activity to less than 1% of wild-type recombinant. In contrast engineering peptide sequences at the C-terminus resulted in specific activities ranging from 0.06–120% of the wild-type recombinant. Addition of cyclic AMP dependent protein kinase catalytic subunit, to a variant luciferase incorporating the kinase recognition sequence, LRRASLG, with a serine at amino-acid position 543 resulted in a 30% reduction in activity. Alkaline phosphatase treatment restored activity. The bioluminescent activity of a variant luciferase containing a thrombin recognition sequence, LVPRES, with the cleavage site positioned between amino acid 542 and 543, decreased by 50% when incubated in the presence of thrombin. The results indicate regions within luciferase where peptide sequences may be engineered while retaining bioluminescent activity and have shown changes in bioluminescent activity when these sites are subjected to covalent modification. Changes in secondary structure, charge and length at the C-terminus of luciferase disrupt the microenvironment of the active site, leading to alterations in light emission. This has important implications both in understanding the evolution of beetle bioluminescence and also in the development of bioluminescent indicators of the covalent modification of proteins.     

Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein.

The entire nucleotide sequence of the rsaA gene, encoding the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A, was determined. The rsaA gene encoded a protein of 1026 amino acids, with a predicted molecular weight of 98,132. Protease cleavage of mature RsaA protein and amino acid sequencing of retrievable peptides yielded two peptides: one aligned with a region approximately two-thirds the way into the predicted amino acid sequence and the second peptide corresponded to the predicted carboxy terminus. Thus, no cleavage processing of the carboxy portion of the RsaA protein occurred during export, and with the exception of the removal of the initial methionine residue, the protein was not processed by cleavage to produce the mature protein. The predicted RsaA amino acid profile was unusual, with small neutral residues predominating. Excepting aspartate, charged amino acids were in relatively low proportion, resulting in an especially acidic protein, with a predicted pI of 3.46. As with most other sequenced S-layer proteins, RsaA contained no cysteine residues. A homology scan of the Swiss Protein Bank 17 produced no close matches to the predicted RsaA sequence. However, RsaA protein shared measurable homology with some exported proteins of other bacteria, including the hemolysins. Of particular interest was a specific region of the RsaA protein that was homologous to the repeat regions of glycine and aspartate residues found in several proteases and hemolysins. These repeats are implicated in the binding of calcium for proper structure and biological activity of these proteins. Those present in the RsaA protein may perform a similar function, since S-layer assembly and surface attachment requires calcium. RsaA protein also shared some homology with 10 other S-layer proteins, with the Campylobacter fetus S-layer protein scoring highest.     

Processing of pro-hormone precursor proteins

Peptide-hormones and other biologically active peptides are synthesized as higher molecular weight precursor proteins (pro-proteins) which must undergo post-translational modification to yield the bioactive peptide(s). These post-translational enzymatic events include limited endoproteolysis and may include other modifications of the generated peptide such as limited exopeptidase digestion, N-terminal acetylation, C-terminal amidation, and formation of N-terminal pyroglutamyl residues (pyrrolation). The secretory vesicle hypothesis, one of the major hypotheses regarding processing, states that the initial endoproteolytic event occurs upon formation of the secretory vesicle (or granule) or within the secretory vesicle from which the bioactive peptides are released. Two different endoproteinases which are likely to be physiologically relevant processing enzymes of pro-atrial natriuretic factor and pro-gonadotropin releasing hormone precursor protein, respectively, have recently been discovered in our laboratory and are discussed as model enzymes in the context of this hypothesis. The results indicate that the precursor protein and its complement of processing enzymes are co-packaged into the secretory granule. Evidence is presented to support the idea that the specific sequence and conformation (secondary structural features) of the processing recognition site within the precursor protein likely contribute in large part to the basis for limited endoproteolysis. In the pro-hormones studied, the recognition site is an extended sequence of five to seven residues which likely exists as a beta-turn at the surface of the precursor protein. By extending our results to appropriate protein sequences in the National Biomedical Research Foundation database, we are suggesting that in addition to the doublet of basic amino acids, the primary processing recognition site in pro-hormone precursor proteins often contains a monobasic amino acid or a strongly polar residue (Glu or Asp) in close sequence proximity to the doublet of basic residues.     

Functional restraints on the patterns of amino acid substitutions: application to sequence-structure homology recognition

Amino acid residues that are involved in functional interactions in proteins have strong evolutionary pressure to remain unchanged and consequently their substitution patterns are different from those that are noninteracting. To characterize and quantify the differences between amino acid substitution patterns due to structural restraints and those under functional restraints, we have made a comparative analysis of families of homologous proteins. Residues classified as having the same amino acid type, secondary structure, accessibility, and side-chain hydrogen bonds are shown to be better conserved if they are close to the active site. We have focused on enzyme families for this analysis since they have functional sites that are easily defined by their catalytic residues. We have derived new sets of environment-specific substitution tables, which we term function-dependent environment-specific substitution tables, where amino acid residues are classified according to their distance from the functional sites. The residues that are within a distance of 9 A from the active site have distinct amino acid substitution patterns when compared to the other sites. The function-dependent environment-specific substitution tables have been tested using the sequence-structure homology recognition program FUGUE and the results compared with the recognition performance obtained using the standard environment-specific substitution tables. Significant improvements are obtained in both recognition performance and alignment accuracy using the function-dependent environment-specific substitution tables (P-value = 0.02, according to the Wilcoxon signed rank test for alignment accuracy). The alignments near the active site are greatly improved with pronounced improvements at lower percentage identities (less than 30%).     

Nucleotide sequence of the cDNA encoding the precursor for mitochondrial serine:pyruvate aminotransferase of rat liver

The nucleotide sequence of the mRNA coding for the precursor of mitochondrial serine:pyruvate aminotransferase of rat liver was determined from those of cDNA clones. The mRNA comprises at least 1533 nucleotides, except the poly(A) tail, and encodes a polypeptide consisting of 414 amino acid residues with a molecular mass of 45,834 Da. Comparison of the N-terminal amino acid sequence of mitochondrial serine:pyruvate aminotransferase with the nucleotide sequence of the mRNA showed that the mature form of the mitochondrial enzyme consisted of 390 amino acid residues of 43,210 Da. The amino acid composition of mitochondrial serine:pyruvate aminotransferase deduced from the nucleotide sequence of the cDNA showed good agreement with the composition determined on acid hydrolysis of the purified protein. The extra 24 amino acid residues correspond to the N-terminal extension peptide (pre-sequence) that is indispensable for the specific import of the precursor protein into mitochondria. In the extension peptide there are four basic amino acids distributed among hydrophobic amino acids and, as revealed on helical wheel analysis, the putative alpha-helical structure of the peptide was amphiphilic in nature. The secondary structures of the mature serine:pyruvate aminotransferase and three other aminotransferases of rat liver were predicted from their amino acid sequences. Their secondary structures exhibited a common feature and so we propose the specific lysine residue which binds pyridoxal phosphate as the active site of serine:pyruvate aminotransferase.     

Characterization of the active site of group B streptococcal hyaluronan lyase

Hyaluronan lyase is secreted by most strains of the human pathogen, group B streptococcus. Site-directed mutagenesis of the enzyme identified three amino acid residues important for enzyme activity, H479, Y488, and R542. These three residues are in close proximity in the putative active site of a homology model of group B streptococcal hyaluronan lyase. The homology model was based on the crystal structure of another related glycosaminoglycan lyase, chondroitin AC lyase, which exhibits different substrate specificity. Two asparagine residues in the active site groove, N429 and N660, were also found to be essential for enzyme activity. In addition, conversion of two adjacent tryptophan residues in the groove to alanines abolished activity. All amino acids found to be essential in GBS hyaluronan lyase are conserved in both enzymes. However, several amino acids in the active site groove of the two enzymes are not conserved. In the 18 cases in which one of these amino acids in GBS hyaluronan lyase was replaced with its corresponding amino acid in chondroitin AC lyase, no major loss of activity or change in substrate specificity was observed.