Mechanism of hyaluronan binding and degradation: structure of Streptococcus pneumoniae hyaluronate lyase in complex with hyaluronic acid disaccharide at 1.7 A resolution

Hyaluronic acid (HA) is an important constituent of the extracellular matrix; its bacterial degradation has been postulated to contribute to the spread of certain streptococci through tissue. Pneumococci and other streptococci produce hyaluronate lyase, an enzyme which depolymerizes HA, thus hyaluronate lyase might contribute directly to bacterial invasion. Although two different mechanisms for lyase action have been proposed, there was no crystallographic evidence to support those mechanisms. Here, we report the high-resolution crystal structure of Streptococcus pneumoniae hyaluronate lyase in the presence of HA disaccharide product, which ultimately provides the first crystallographic evidence for the binding of HA to hyaluronate lyase. This structural complex revealed a key interaction between the Streptococcus peneumoniae hyaluronate lyase protein and the product, and supports our previously proposed novel catalytic mechanism for HA degradation based on the native Streptococcus peneumoniae hyaluronate lyase structure. The information provided by this complex structure will likely be useful in the development of antimicrobial pharmaceutical agents.     

Genome-based identification of a carbohydrate binding module in Streptococcus pneumoniae hyaluronate lyase

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the connective tissues of higher animals, thereby destroying the normal connective tissue structure and exposing the host tissue cells to various endo- and exogenous factors, including bacterial toxins. The 3D crystal structures of functionally active but truncated Streptococcus pneumoniae and S. agalactiae hyaluronate lyases, along with their substrate and product complexes, have been determined. The enzymes are multidomain proteins with helical barrel-like catalytic domains and two types of beta-sheet domains. Here, through genome-based bioinformatics studies we identify an additional beta-sheet domain present in the most N-terminal part of streptococcal hyaluronate lyases. Fold recognition and modeling studies show that the domain is structurally similar to carbohydrate binding modules and is therefore likely to be directly involved in hyaluronan binding. Likely carbohydrate binding residues were identified and electrostatic complementarity of the hyaluronate lyase domain with hyaluronan demonstrated. The newly identified presumed hyaluronan binding domain likely improves catalytic efficiency by colocalizing the enzyme and its substrate. Other possible functions are discussed. Two contacting aromatic residues are conserved in the hydrophobic core of the hyaluronate lyase domain and in many, perhaps all, families in the superfamily in which they may be placed. This observation may help the identification and classification of other carbohydrate binding modules.     

Kinetic properties of Streptococcus pneumoniae hyaluronate lyase

Streptococcus pneumoniae hyaluronate lyase is a surface antigen of this bacterial pathogen, which causes significant mortality and morbidity in human populations worldwide. The primary function of this enzyme is the degradation of hyaluronan, a major component of the extracellular matrix of the tissues of practically all vertebrates. The enzyme uses a processive mode of action to degrade hyaluronan to a final product, an unsaturated disaccharide hyaluronan unit. This catalysis proceeds via a five-step proton acceptance and donation mechanism that includes substrate binding, catalysis, release of the disaccharide product, translocation of the remaining hyaluronan substrate, and proton exchange with microenvironment. Based on the analysis of the three-dimensional structure of the native enzyme and its complexes with hexasaccharide substrate and disaccharide product, several residues have been chosen for mutation studies. These mutated residues included the catalytic residues Asn349, His399, Tyr408, and residues responsible for substrate binding and translocation, Arg243 and Asn580. The comparison of the kinetic properties of the wild-type with the mutant enzymes allowed for the characterization of every mutant and the correlation of the kinetic properties of the enzyme with its structure. The comparison of the wild-type hyaluronate lyase with other polysaccharide-degrading enzymes, the hydrolases endonuclease and glucoamylase, shows striking similarity of K(m)s for all of these different enzymes.     

Hyaluronan binding and degradation by Streptococcus agalactiae hyaluronate lyase

Streptococcus agalactiae hyaluronate lyase is a virulence factor that helps this pathogen to break through the biophysical barrier of the host tissues by the enzymatic degradation of hyaluronan and certain chondroitin sulfates at beta-1,4 glycosidic linkages. Crystal structures of the native enzyme and the enzyme-product complex were determined at 2.1- and 2.2-A resolutions, respectively. An elongated cleft transversing the middle of the molecule has been identified as the substrate-binding place. Two product molecules of hyaluronan degradation were observed bound to the cleft. The enzyme catalytic site was identified to comprise three residues: His(479), Tyr(488), and Asn(429). The highly positively charged cleft facilitates the binding of the negatively charged polymeric substrate chain. The matching between the aromatic patch of the enzyme and the hydrophobic patch of the substrate chain anchors the substrate chain into degradation position. A pair of proton exchanges between the enzyme and the substrate results in the cleavage of the beta-1,4 glycosidic linkage of the substrate chain and the unsaturation of the product. Phe(423) likely determines the size of the product at the product release side of the catalytic region. Hyaluronan chain is processively degraded from the reducing end toward the nonreducing end. The unsulfated or 6-sulfated regions of chondroitin sulfate can also be degraded in the same manner as hyaluronan.     

Mechanism of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase. Structures of complexes with the substrate

Hyaluronate lyase enzymes degrade hyaluronan, the main polysaccharide component of the host connective tissues, predominantly into unsaturated disaccharide units, thereby destroying the normal connective tissue structure and exposing the tissue cells to various endo- and exogenous factors, including bacterial toxins. The crystal structures of Streptococcus pneumoniae hyaluronate lyase with tetra- and hexasaccharide hyaluronan substrates bound in the active site were determined at 1.52- and 2.0-A resolution, respectively. Hexasaccharide is the longest substrate segment that binds entirely within the active site of these enzymes. The enzyme residues responsible for substrate binding, positioning, catalysis, and product release were thereby identified and their specific roles characterized. The involvement of three residues in catalysis, Asn(349), His(399), and Tyr(408), is confirmed, and the details of proton acceptance and donation within the catalytic machinery are described. The mechanism of processivity of the enzyme is analyzed. The flexibility (allosteric) behavior of the enzyme may be understood in terms of the results of flexibility analysis of this protein, which identified two modes of motion that are also proposed to be involved in the hyaluronan degradation process. The first motion describes an opening and closing of the catalytic cleft located between the alpha- and beta-domains. The second motion demonstrates the mobility of a binding cleft, which may facilitate the binding of the negatively charged hyaluronan to the enzyme.     

Structure and flexibility of Streptococcus agalactiae hyaluronate lyase complex with its substrate. Insights into the mechanism of processive degradation of hyaluronan

Streptococcus agalactiae hyaluronate lyase degrades primarily hyaluronan, the main polysaccharide component of the host connective tissues, into unsaturated disaccharide units as the end product. Such function of the enzyme destroys the normal connective tissue structure of the host and exposes the tissue cells to various bacterial toxins. The crystal structure of hexasaccharide hyaluronan complex with the S. agalactiae hyaluronate lyase was determined at 2.2 A resolution; the mechanism of the catalytic process, including the identification of specific residues involved in the degradation of hyaluronan, was clearly identified. The enzyme is composed structurally and functionally from two distinct domains, an alpha-helical alpha-domain and a beta-sheet beta-domain. The flexibility of the protein was investigated by comparing the crystal structures of the S. agalactiae and the Streptococcus pneumoniae enzymes, and by using essential dynamics analyses of CONCOORD computer simulations. These revealed important modes of flexibility, which could be related to the protein function. First, a rotation/twist of the alpha-domain relative to the beta-domain is potentially related to the mechanism of processivity of the enzyme; this twist motion likely facilitates shifting of the ligand along the catalytic site cleft in order to reposition it to be ready for further cleavage. Second, a movement of the alpha- and beta-domains with respect to each other was found to contribute to a change in electrostatic characteristics of the enzyme and appears to facilitate binding of the negatively charged hyaluronan ligand. Third, an opening/closing of the substrate binding cleft brings a catalytic histidine closer to the cleavable substrate beta1,4-glycosidic bond. This opening/closing mode also reflects the main conformational difference between the crystal structures of the S. agalactiae and the S. pneumoniae hyaluronate lyases.     

Structural basis of hyaluronan degradation by Streptococcus pneumoniae hyaluronate lyase

Streptococcus pneumoniae hyaluronate lyase (spnHL) is a pathogenic bacterial spreading factor and cleaves hyaluronan, an important constituent of the extra- cellular matrix of connective tissues, through an enzymatic beta-elimination process, different from the hyaluronan degradation by hydrolases in animals. The mechanism of hyaluronan binding and degradation was proposed based on the 1.56 A resolution crystal structure, substrate modeling and mutagenesis studies on spnHL. Five mutants, R243V, N349A, H399A, Y408F and N580G, were constructed and their activities confirmed our mechanism hypothesis. The important roles of Tyr408, Asn349 and His399 in enzyme catalysis were proposed, explained and confirmed by mutant studies. The remaining weak enzymatic activity of the H399A mutant, the role of the free carboxylate group on the glucuronate residue, the enzymatic behavior on chondroitin and chondroitin sulfate, and the small activity increase in the N580G mutant were explained based on this mechanism. A possible function of the C-terminal beta-sheet domain is to modulate enzyme activity through binding to calcium ions.     

Structures of Streptococcus pneumoniae hyaluronate lyase in complex with chondroitin and chondroitin sulfate disaccharides. Insights into specificity and mechanism of action

Streptococcus pneumoniae hyaluronate lyase is a surface enzyme of this Gram-positive bacterium. The enzyme degrades hyaluronan and chondroitin/chondroitin sulfates by cleaving the beta1,4-glycosidic linkage between the glycan units of these polymeric substrates. This degradation helps spreading of this bacterial organism throughout the host tissues and facilitates the disease process caused by pneumococci. The mechanism of this degradative process is based on beta-elimination, is termed proton acceptance and donation, and involves selected residues of a well defined catalytic site of the enzyme. The degradation of hyaluronan alone is thought to proceed through a processive mode of action. The structures of complexes between the enzyme and chondroitin as well as chondroitin sulfate disaccharides allowed for the first detailed insights into these interactions and the mechanism of action on chondroitins. This degradation of chondroitin/chondroitin sulfates is nonprocessive and is selective for the chondroitin sulfates only with certain sulfation patterns. Chondroitin sulfation at the 4-position on the nonreducing site of the linkage to be cleaved or 2-sulfation prevent degradation due to steric clashes with the enzyme. Evolutionary studies suggest that hyaluronate lyases evolved from chondroitin lyases and still retained chondroitin/chondroitin sulfate degradation abilities while being specialized in the degradation of hyaluronan. The more efficient processive degradation mechanism has come to be preferred for the unsulfated substrate hyaluronan.     

Crystal structures of a family 8 polysaccharide lyase reveal open and highly occluded substrate-binding cleft conformations

Bacterial enzymatic degradation of glycosaminoglycans such as hyaluronan and chondroitin is facilitated by polysaccharide lyases. Family 8 polysaccharide lyase (PL8) enzymes contain at least two domains: one predominantly composed of α-helices, the α-domain, and another predominantly composed of β-sheets, the β-domain. Simulation flexibility analyses indicate that processive exolytic cleavage of hyaluronan, by PL8 hyaluronate lyases, is likely to involve an interdomain shift, resulting in the opening/closing of the substrate-binding cleft between the α- and β-domains, facilitating substrate translocation. Here, the Streptomyces coelicolor A3(2) PL8 enzyme was recombinantly expressed in and purified from Escherichia coli and biochemically characterized as a hyaluronate lyase. By using X-ray crystallography its structure was solved in complex with hyaluronan and chondroitin disaccharides. These findings show key catalytic interactions made by the different substrates, and on comparison with all other PL8 structures reveals that the substrate-binding cleft of the S. coelicolor enzyme is highly occluded. A third structure of the enzyme, harboring a mutation of the catalytic tyrosine, created via site-directed mutagenesis, interestingly revealed an interdomain shift that resulted in the opening of the substrate-binding cleft. These results add further support to the proposed processive mechanism of action of PL8 hyaluronate lyases and may indicate that the mechanism of action is likely to be universally used by PL8 hyaluronate lyases.     

Unusual structural features of the bacteriophage-associated hyaluronate lyase (hylp2)

Hyaluronate lyases are a class of endoglycosaminidase enzymes, which are of considerable complexity and heterogeneity. Their primary function is to degrade hyaluronan and certain other glycosaminoglycans and facilitate the spread of disease. Among hyaluronate lyases, the bacteriophage-associated enzymes are unique as they have the lowest molecular mass, very low amino acid sequence homology with bacterial hyaluronate lyases, and exhibit absolute specificity for one type of glycosaminoglycan, i.e. hyaluronan. Despite such unique characteristics significant details on structural features of these lyases are not available. The Streptococcus pyogenes bacteriophage 10403 contains a gene, hylP2, which encodes for hyaluronate lyase (HylP2) in this organism. HylP2 was cloned, overexpressed, and purified to homogeneity. The recombinant HylP2 exists as a homotrimer of molecular mass about 110 kDa, under physiological conditions. Limited proteolysis and guanidine hydrochloride denaturation studies demonstrated that the N-terminal region of the protein is flexible, whereas the C-terminal portion has a compact conformation. The enzyme shows sequential unfolding, with the N-terminal unfolding first followed by the simultaneous unfolding and dissociation of the stabilized trimeric C-terminal domain. We isolated a functionally active C-terminal fragment (Ser(128)-Lys(337)) of the protein that was stabilized in a trimeric configuration. Comparative functional studies with full-length protein, N:C complex, and isolated C-terminal domain demonstrated that the active site of HylP2 is present in the C-terminal portion of the enzyme, and the N-terminal portion modulates the substrate specificity and enzymatic activity of the C-terminal domain.     

The function of hydrophobic residues in the catalytic cleft of Streptococcus pneumoniae hyaluronate lyase. Kinetic characterization of mutant enzyme forms

Streptococcus pneumoniae hyaluronate lyase is a surface antigen of this Gram-positive human bacterial pathogen. The primary function of this enzyme is the degradation of hyaluronan, which is a major component of the extracellular matrix of the tissues of vertebrates and of some bacteria. The enzyme degrades its substrate through a beta-elimination process called proton acceptance and donation. The inherent part of this degradation is a processive mode of action of the enzyme degrading hyaluronan into unsaturated disaccharide hyaluronic acid blocks from the reducing to the nonreducing end of the polymer following the initial random endolytic binding to the substrate. The final degradation product is the unsaturated disaccharide hyaluronic acid. The residues of the enzyme that are involved in various aspects of such degradation were identified based on the three-dimensional structures of the native enzyme and its complexes with hyaluronan substrates of various lengths. The catalytic residues were identified to be Asn(349), His(399), and Tyr(408). The residues responsible for the release of the product of the reaction were identified as Glu(388), Asp(398), and Thr(400), and they were termed negative patch. The hydrophobic residues Trp(291), Trp(292), and Phe(343) were found to be responsible for the precise positioning of the substrate for enzyme catalysis and named hydrophobic patch. The comparison of the specific activities and kinetic properties of the wild type and the mutant enzymes involving the hydrophobic patch residues W292A, F343V, W291A/W292A, W292A/F343V, and W291A/W292A/F343V allowed for the characterization of every mutant and for the correlation of the activity and kinetic properties of the enzyme with its structure as well as the mechanism of catalysis.     

Streptococcus pneumoniae hyaluronate lyase contains two non-cooperative independent folding/unfolding structural domains: characterization of functional domain and inhibitors of enzyme

Hyaluronate lyase contributes directly to bacterial invasion by degrading hyaluronan, the major component of host extracellular matrix of connective tissues. Streptococcus pneumoniae hyaluronate lyase (SpnHL) is built from two structural domains that interact through interface residues, in addition to being connected by a peptide linker. For the first time we demonstrate that the N- and C-terminal domains of SpnHL fold/unfold independent of each other suggesting the absence of any significant cooperative interactions between them. The C-terminal domain of SpnHL is less stable than the N-terminal domain against thermal and guanidine hydrochloride denaturation. The intact N-terminal domain was purified after limited proteolysis of SpnHL under conditions where only the C-terminal domain was unfolded. Isolated N-terminal domain of SpnHL had similar thermal stability as when present in the native enzyme and was found to be enzymatically active demonstrating that it is capable of carrying out enzymatic reaction on its own. Functional studies demonstrated that guanidine hydrochloride, guanidine isothiocyanate, l-arginine methyl ester, and l-arginine inhibit the enzymatic activity of SpnHL at very low concentrations. This provides a lead for new chemical entities that can be exploited for designing effective inhibitors of SpnHL.     

Domain motions of hyaluronan lyase underlying processive hyaluronan translocation

Hyaluronan lyase (Hyal) is a surface enzyme occurring in many bacterial organisms including members of Streptococcus species. Streptococcal Hyal primarily degrades hyaluronan‐substrate (HA) of the extracellular matrix. This degradation appears to facilitate the spread of this bacterium throughout host tissues. Unlike purely endolytic degradation of its other substrates, unsulfated chondroitin or some chondroitin sulfates, the degradation of HA by Hyal proceeds by processive exolytic cleavage of one disaccharide at a time following an initial endolytic cut. Molecular dynamics (MD) studies of Hyal from Streptococcus pneumoniae are presented that address the enzyme's molecular mechanism of action and the role of domain motions for processive functionality. The analysis of extensive sub‐microsecond MD simulations of this enzyme action on HA‐substrates of different lengths and the connection between the domain dynamics of Hyal and the translocation of the HA‐substrate reveals that opening/closing and twisting domain motions of the Hyal are intimately linked to processive HA degradation. Enforced simulations confirmed this finding as the domain motions in SpnHyal were found to be induced by enforced substrate translocation. These results establish the dynamic interplay between Hyal flexibility and substrate translocation and provide insight into the processive mechanism of Hyal. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Effects of Ascorbic Acid and Analogs on the Activity of Testicular Hyaluronidase and Hyaluronan Lyase on Hyaluronan

We have evaluated the inhibition of testicular hyaluronidase and hyaluronan lyase by L-ascorbic acid and chemical analogs. We observed that L-ascorbic acid, D-isoascorbic acid and dehydroascorbic acid inhibited both types of enzymes, but showed stronger effects towards hyaluronan lyase. But these compounds were observed to degrade the substrate, hyaluronan, by themselves. Of the other ascorbic acid analogs tested, saccharic acid inhibited hyaluronan lyase, while not affecting the enzymatic activity of testicular hyaluronidase, nor affecting the physic-chemical stability of hyaluronan. This is the first compound, to our knowledge, to be shown to possess such selective inhibition. Therefore, we propose that saccharic acid could serve as a lead compound for the development of potent and selective inhibitors of bacterial hyaluronan lyase or of polysaccharide lyase enzymes in general as we observed this compound to be capable of inhibiting chondroitinase ABC in addition to hyaluronan lyase.     

Effects of ascorbic acid and analogs on the activity of testicular hyaluronidase and hyaluronan lyase on hyaluronan

We have evaluated the inhibition of testicular hyaluronidase and hyaluronan lyase by L-ascorbic acid and chemical analogs. We observed that L-ascorbic acid, D-isoascorbic acid and dehydroascorbic acid inhibited both types of enzymes, but showed stronger effects towards hyaluronan lyase. But these compounds were observed to degrade the substrate, hyaluronan, by themselves. Of the other ascorbic acid analogs tested, saccharic acid inhibited hyaluronan lyase, while not affecting the enzymatic activity of testicular hyaluronidase, nor affecting the physic-chemical stability of hyaluronan. This is the first compound, to our knowledge, to be shown to possess such selective inhibition. Therefore, we propose that saccharic acid could serve as a lead compound for the development of potent and selective inhibitors of bacterial hyaluronan lyase or of polysaccharide lyase enzymes in general as we observed this compound to be capable of inhibiting chondroitinase ABC in addition to hyaluronan lyase.     

The signal-to-noise ratio as a measure of HA oligomer concentration: a MALDI-TOF MS study

MALDI-TOF MS (matrix-assisted laser desorption and ionization time-of-flight mass spectrometry) was used to determine ng amounts of defined hyaluronan (HA) oligomers obtained by enzymatic digestion of high molecular weight HA with testicular hyaluronate lyase. The signal-to-noise (S/N) ratio of the positive and negative ion spectra represents a reliable concentration measure: Amounts of HA down to about 40 fmol could be determined and there is a linear correlation between the S/N ratio and the HA amount between about 0.8 pmol and 40 fmol. However, the detection limits depend considerably on the size of the HA oligomer with larger oligomers being less sensitively detectable. The advantages and drawbacks of the S/N ratio as concentration measure are discussed.     

Efficient synthesis of an aldehyde functionalized hyaluronic acid and its application in the preparation of hyaluronan-lipid conjugates

An efficient method to synthesize hyaluronan oligosaccharide lipid conjugates is described. This strategy is based on the introduction of a double bond in the glucuronic acid of the hyaluronic acid (HA), by the biodegradation of HA with hyaluronate lyase, followed by the generation of a free aldehyde group at the nonreducing end of hyaluronic acid via ozonolysis and the subsequent reduction of the generated ozonide. The resulting aldehyde-functionalized HA is then coupled to dipalmitoyl phosphatidylethanolamine (DPPE) using reductive amination chemistry. This methodology can be extended to link molecules such as biotin, polymers, or proteins to HA for numerous applications in drug delivery and in the creation of biocompatible materials for tissue repair and engineering.     

Modulation of hyaluronan in the migratory pathway of mouse primordial germ cells

In the present report we followed the distribution of hyaluronan during the phases of separation, migration, and colonization of the primordial germ cell migratory process. Hyaluronan was detected by the use of two cytochemical methods: (1) ruthenium hexammine trichloride (RHT) associated with enzymatic treatment with hyaluronate lyase and (2) a binding specific probe for hyaluronan. After RHT treatment the proteoglycans and/or glycosaminoglycans were observed as a meshwork formed by electron-dense granules connected by thin filaments. After enzymatic digestion, no filaments could be detected in the migratory pathway. Quantitative analysis showed a close correlation between cell migration and the concentration of RHT-positive filaments. It was also shown that high amounts of hyaluronan were expressed in the separation phase and migration phases whereas during the colonization phase the amount of hyaluronan was clearly diminished. This study showed that the presence of primordial germ cells in each compartment of the migratory pathway was always accompanied by a high expression of hyaluronan. These results indicate that hyaluronan is an important molecule in the migratory process, providing the primordial germ cells with a hydrated environment that facilitates their movement toward the genital ridges.     

Complementary exploration of the action pattern of hyaluronate lyase from Streptococcus agalactiae using capillary electrophoresis, gel-permeation chromatography and viscosimetric measurements

Hyaluronic acid (HA) was treated with hyaluronate lyase (GBS HA lyase, E.C. 4.2.2.1, from Streptococcus agalactiae strain 4755), and the products have been analyzed by capillary electrophoresis (CE-UV and online CE-ESIMS), gel-permeation chromatography (GPC) and viscosimetric measurements. The resulting electropherograms showed that the enzyme produced a mixture of oligosaccharides with a 4,5-unsaturated uronic acid nonreducing terminus. More exhaustive degradation of HA led to increasing amounts of di-, tetra-, hexa-, octa- and decasaccharides. Using CE, linear relationships were found between peak area of the observed oligosaccharides and reaction time. Determination of viscosity at different stages of reaction yielded an initial rapid decrease following Michaelis-Menten theory. A reaction time-dependent change in the elution position of the HA peak due to partial digestion of HA with GBS hyaluronate lyase has been observed by GPC. These results indicated that the HA lyase under investigation is an eliminase that acts in a nonprocessive endolytic manner, as at all stages of digestion a mixture of oligosaccharides of different size were found. For GBS HA lyase from Streptococcus agalactiae strain 3502, previously published findings reported an action pattern that involves an initial random endolytic cleavage followed by rapid exolytic and processive release of unsaturated disaccharides. Our results suggest that differences between the two enzymes from distinct S. agalactiae strains (GBS strains 4755 and 3502) have to be considered.     

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.