Kinetic Characterization of the Recombinant Hyaluronan Synthases from Streptococcus pyogenes and Streptococcus equisimilis
1999; Elsevier BV; Volume: 274; Issue: 7 Linguagem: Inglês
10.1074/jbc.274.7.4246
ISSN1083-351X
AutoresValarie L. Tlapak‐Simmons, Bruce A. Baggenstoss, Kshama Kumari, Coy D. Heldermon, Paul H. Weigel,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoThe two hyaluronan synthases (HASs) fromStreptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as recombinant proteins containing His6 tails. The accompanying paper has described the purification and lipid dependence of both HASs, their preference for cardiolipin, and their stability during storage (Tlapak-Simmons, V. L., Baggenstoss, B. A., Clyne, T., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4239–4245). Kinetic characterization of the enzymes in isolated membranes gave K m values for UDP-GlcUA of 40 ± 4 μm for spHAS and 51 ± 5 μm for seHAS. In both cases, theV max profiles at various concentrations of UDP-GlcNAc were hyperbolic, with no evidence of cooperativity. In contrast, membrane-bound spHAS, but not seHAS, showed sigmoidal behavior as the UDP-GlcNAc concentration was increased, with a Hill number of ∼2, indicating significant cooperativity. The Hill number for UDP-GlcNAc utilization by seHAS was 1, confirming the lack of cooperativity for UDP-GlcNAc in this enzyme. The K mvalues for UDP-GlcNAc were 60 ± 7 μm for seHAS and 149 ± 3 μm for spHAS in the isolated membranes. The kinetic characteristics of the two affinity-purified HAS enzymes were assessed in the presence of cardiolipin after 8–9 days of storage at –80 °C without cardiolipin. With increasing storage time, the enzymes showed a gradual increase in their K mvalues for both substrates and a decrease inV max. Even in the presence of cardiolipin, the detergent-solubilized, purified HASs had substantially higherK m values for both substrates than the membrane-bound enzymes. The K UDP-GlcUA for purified spHAS and seHAS increased 2–4-fold. TheK UDP-GlcNAc for spHAS and seHAS increased 4- and 5-fold, respectively. Despite the higher K mvalues, the V max values for the purified HASs were only ∼50% lower than those for the membrane-bound enzymes. Significantly, purified spHAS displayed the same cooperative interaction with UDP-GlcNAc (n H ∼ 2), whereas purified seHAS showed no cooperativity. The two hyaluronan synthases (HASs) fromStreptococcus pyogenes (spHAS) and Streptococcus equisimilis (seHAS) were expressed in Escherichia coli as recombinant proteins containing His6 tails. The accompanying paper has described the purification and lipid dependence of both HASs, their preference for cardiolipin, and their stability during storage (Tlapak-Simmons, V. L., Baggenstoss, B. A., Clyne, T., and Weigel, P. H. (1999) J. Biol. Chem. 274, 4239–4245). Kinetic characterization of the enzymes in isolated membranes gave K m values for UDP-GlcUA of 40 ± 4 μm for spHAS and 51 ± 5 μm for seHAS. In both cases, theV max profiles at various concentrations of UDP-GlcNAc were hyperbolic, with no evidence of cooperativity. In contrast, membrane-bound spHAS, but not seHAS, showed sigmoidal behavior as the UDP-GlcNAc concentration was increased, with a Hill number of ∼2, indicating significant cooperativity. The Hill number for UDP-GlcNAc utilization by seHAS was 1, confirming the lack of cooperativity for UDP-GlcNAc in this enzyme. The K mvalues for UDP-GlcNAc were 60 ± 7 μm for seHAS and 149 ± 3 μm for spHAS in the isolated membranes. The kinetic characteristics of the two affinity-purified HAS enzymes were assessed in the presence of cardiolipin after 8–9 days of storage at –80 °C without cardiolipin. With increasing storage time, the enzymes showed a gradual increase in their K mvalues for both substrates and a decrease inV max. Even in the presence of cardiolipin, the detergent-solubilized, purified HASs had substantially higherK m values for both substrates than the membrane-bound enzymes. The K UDP-GlcUA for purified spHAS and seHAS increased 2–4-fold. TheK UDP-GlcNAc for spHAS and seHAS increased 4- and 5-fold, respectively. Despite the higher K mvalues, the V max values for the purified HASs were only ∼50% lower than those for the membrane-bound enzymes. Significantly, purified spHAS displayed the same cooperative interaction with UDP-GlcNAc (n H ∼ 2), whereas purified seHAS showed no cooperativity. HA 1The abbreviations used are: HA, hyaluronan or hyaluronic acid; HAS, hyaluronan synthase; seHAS, S. equisimilis hyaluronan synthase; spHAS, S. pyogeneshyaluronan synthase; CL, cardiolipin.1The abbreviations used are: HA, hyaluronan or hyaluronic acid; HAS, hyaluronan synthase; seHAS, S. equisimilis hyaluronan synthase; spHAS, S. pyogeneshyaluronan synthase; CL, cardiolipin. is a polysaccharide composed of two alternating sugars, β1,3-linked glucuronic acid and β1,4-linked N-acetylglucosamine (1Meyer K. Palmer J.W. J. Biol. Chem. 1934; 107: 629-634Abstract Full Text PDF Google Scholar). Although the structure of HA seems quite simple, the molecule, nonetheless, has unusual physical properties that are important for its numerous biological functions (2Evered D. Whelan J. CIBA Found. Symp. 1989; 143: 1-288Google Scholar, 3Laurent T.C. Acta Oto-laryngol. 1987; 442: 7-24Crossref Scopus (217) Google Scholar, 4Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1242Crossref PubMed Scopus (599) Google Scholar, 5Toole B.P. Hay E.D. Cell Biology of the Extracellular matrix. Plenum Press, New York1991: 305-341Google Scholar). For example, HA forms very viscous solutions and gels due to its high molecular mass and its ability to bind cations and to hydrate large amounts of water. This characteristic of HA provides the viscous lubrication of synovial fluid and helps provide cartilage with its viscoelasticity. These characteristics are also ideal for the role HA has in the extracellular matrices (4Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1242Crossref PubMed Scopus (599) Google Scholar, 5Toole B.P. Hay E.D. Cell Biology of the Extracellular matrix. Plenum Press, New York1991: 305-341Google Scholar) of the skin and virtually every vertebrate tissue as well as in the fluid of the vitreous humor of the eye. HA also plays an important role in morphogenesis, wound healing (6Abatangelo G. Martinelli M. Vecchia P. J. Surg. Res. 1983; 35: 410-416Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 7Balazs E.A. Denlinger J.L. J. Rheumatol. 1993; 20: 3-9Google Scholar, 8Goa K.L. Benfield P. Drugs. 1994; 47: 536-566Crossref PubMed Scopus (379) Google Scholar, 9Gressner A.M. Bachem M.G. Digestion. 1995; 56: 335-346Crossref PubMed Scopus (222) Google Scholar), and angiogenesis (10West D.C. Hampson I.N. Arnold F. Kumars S. Science. 1985; 228: 1324-1326Crossref PubMed Scopus (961) Google Scholar, 11Feinberg R.N. Beebe D.C. Science. 1983; 220: 1177-1179Crossref PubMed Scopus (299) Google Scholar). HA receptors and HA-binding proteins, particularly CD44 (12Lesley J. Hyman R. Kincade P.W. Adv. Immunol. 1993; 54: 271-335Crossref PubMed Scopus (1026) Google Scholar) and the receptor for hyaluronic acid-mediated mobility (RHAMM; Ref. 13Turley E.A. Bowman P. Kytryk M.A. J. Cell Sci. 1985; 78: 133-145PubMed Google Scholar), modulate cellular responses to HA.The first HAS gene to be cloned was from Group A Streptococcus pyogenes (14DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 14568-14571Abstract Full Text PDF PubMed Google Scholar, 15DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar, 16DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (96) Google Scholar). When the bona fide HAS from Group CStreptococcus equisimilis was later cloned (17Kumari K. Weigel P.H. J. Biol. Chem. 1997; 272: 32539-32546Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), the seHAS protein showed 70 and 72% identities to spHAS at the nucleotide and amino acid sequence levels, respectively. After discovery of the spHAS gene, a related family of homologous cDNAs and enzymes was then found in eukaryotes (18Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar, 19Spicer A.P. McDonald J.A J. Biol. Chem. 1998; 273: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). These HASs include human HAS1 and HAS2 (20Itano N. Kimata K. Biochem. Biophys. Res. Commun. 1996; 222: 816-820Crossref PubMed Scopus (104) Google Scholar, 21Shyjan A.M. Heldin P. Butcher E.C. Yoshino T. Briskin M.J. J. Biol. Chem. 1996; 271: 23395-23399Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 22Watanabe K. Yamaguchi Y. J. Biol. Chem. 1996; 271: 22945-22948Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar); murine HAS1, HAS2, and HAS3 (19Spicer A.P. McDonald J.A J. Biol. Chem. 1998; 273: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 23Itano N. Kimata K. J. Biol. Chem. 1996; 271: 9875-9878Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 24Spicer A.P. Augustine M.L. McDonald J.A. J. Biol. Chem. 1996; 271: 23400-23406Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar); chicken HAS2 and HAS3 (19Spicer A.P. McDonald J.A J. Biol. Chem. 1998; 273: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar); and Xenopus laevis HAS1, HAS2, HAS3, and HAS-related sequence (19Spicer A.P. McDonald J.A J. Biol. Chem. 1998; 273: 1923-1932Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 25DeAngelis P.L. Achyuthan A.M. J. Biol. Chem. 1996; 271: 23657-23660Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 26Meyer M.F. Kreil G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4543-4547Crossref PubMed Scopus (88) Google Scholar, 27Rosa F. Sargent T.D. Rebbert M.L. Micheals G.S. Jamrich M. Grunz H. Jonas E. Winkels J.A. Dawid I.B. Dev. Biol. 1988; 129: 114-123Crossref PubMed Scopus (85) Google Scholar, 28Semino C.E. Robins P.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3498-3501Crossref PubMed Scopus (73) Google Scholar, 29Semino A.M. Specht A.A. Raimondi A. Robbins P.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 47-53Crossref Scopus (94) Google Scholar). In addition, HASs have also been identified and cloned (30DeAngelis P.L. Jing W. Graves M.V. Burbank D.E. Van Etten J.L. Science. 1997; 278: 1800-1803Crossref PubMed Scopus (108) Google Scholar) from chlorella virus PBCV-1 (A98R) and fromPasteurella multocida (31DeAngelis P.L. Jing W. Drake R.R. Achyuthan A.M. J. Biol. Chem. 1998; 273: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Although the two streptococcal HASs are very similar, the HAS from P. multocida is quite different structurally. Similarities among the prokaryotic and eukaryotic members of this HAS family have been reviewed (18Weigel P.H. Hascall V.C. Tammi M. J. Biol. Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). These various HAS enzymes comprise a large family of proteins with many common features and regions of amino acid sequence identity or similarity.To understand the important role of the HA polysaccharide in normal development and health and in various diseases, it is critical to know more about the HASs, the enzymes responsible for HA synthesis. We need to know how these HASs work to assemble the HA polymer and how the enzymes are regulated. In the accompanying paper (32Tlapak-Simmons V.L. Baggenstoss B.A. Clyne T. Weigel P.H. J. Biol. Chem. 1999; 274: 4239-4245Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), we reported the purification and lipid dependence of active recombinant spHAS and seHAS expressed in Escherichia coli. In the present study, we have determined, for the first time in the absence of other streptococcal proteins or factors, the kinetic constants for both HAS enzymes in membranes and after detergent solubilization and purification. A preliminary report of these findings was reported earlier (33Tlapak-Simmons V.L. Baggenstoss B.A. Weigel P.H. Glycobiology. 1997; 7: 1032Google Scholar).DISCUSSIONHistorically, the first cell-free studies of HA biosynthesis used Group A streptococcal bacteria. The pioneering work of Dorfman and co-workers (39Markovitz A. Cifonelli J.A. Dorfman A. J. Biol. Chem. 1959; 234: 2343-2350Abstract Full Text PDF PubMed Google Scholar, 44Stoolmiller A.C. Dorfman A. J. Biol. Chem. 1969; 244: 236-246Abstract Full Text PDF PubMed Google Scholar) in the 1950s and 1960s showed that the streptococcal HAS was located in the cell membrane, required Mg2+ ions, and used the two sugar nucleotide substrates UDP-GlcUA and UDP-GlcNAc to polymerize a HA chain. Subsequently, however, these and many other workers were unable to solubilize the enzyme in an active and stable form or to purify it. Similarly, eukaryotic HASs have not yet been purified. Isolation of the Group A and Group C streptococcal HAS genes has now allowed us to express these proteins in large amounts in E. coli (32Tlapak-Simmons V.L. Baggenstoss B.A. Clyne T. Weigel P.H. J. Biol. Chem. 1999; 274: 4239-4245Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).All of the known enzymes catalyze reactions that use one or two (or, rarely, three) substrates and produce one or two products. HASs are unique among the enzymes characterized to date. The HAS has two different enzyme activities (i.e. glycosyltransferases) in the same protein, and the HA product after each sugar addition becomes the acceptor for the next sugar addition. HASs are also membrane enzymes. The overall reaction for the synthesis of one HA disaccharide unit is shown in Equation 1, UDP-GlcUA+UDP-GlcNAc+(HA)n→HAS(HA)n+1+2UDPEquation 1 where n is the number of disaccharide units. Although it seems straightforward, the enzyme must possess at least six (and probably seven) different functions to perform this overall reaction, as shown in Fig. 6. Numerous questions about the details and mechanism of this complex reaction can be answered now that the streptococcal HAS enzymes have been purified.HAS enzymes are unique in an additional respect because the two sugar nucleotide substrates are structurally so similar, they each have the possibility of competing with the other for the appropriate UDP-sugar-binding site on the enzyme. This cross-talk hypothesis is illustrated in Fig. 6 by the dashed lines that show, for example, UDP-GlcUA interacting with the UDP-GlcNAc-binding site. Although the HASs do not misincorporate other sugar nucleotides into the growing HA chain, other UDP-sugars may transiently occupy a binding site and thus be competitive inhibitors. Initial experiments have indicated that 0–1 mm concentrations of sugar nucleotides like UDP-Glc or UDP-GalUA or even UDP alone decrease the rate of HA synthesis by spHAS or seHAS. 2K. Kumari, V. L. Tlapak-Simmons, and P. H. Weigel, manuscript in preparation. With high concentrations of one or both of the correct substrates, we observed enzyme inhibition in the presence of a third sugar nucleotide. Even with just the two normal substrates, the rate of HA synthesis becomes biphasic if the concentration of one UDP-sugar is much greater than the other (e.g. Fig. 2 A at a ratio of 100:1). There are no previous reports of this cross-talk phenomenon affecting HA biosynthesis. Observation of this kinetic behavior reflects the advantage of studying purified HAS free from other sugar nucleotide-binding proteins or glycosyltransferases.The scheme in Fig. 6 does not indicate whether sugars are added to the growing HA chain individually or in a coordinated manner as a disaccharide unit as proposed by Saxena et al. (40Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar) for β-glycosyltransferases predicted to contain two functional domains by hydrophobic cluster analysis. Our present kinetic data for the streptococcal HASs do not allow discrimination between these two models. The model of Saxena et al. (40Saxena I.M. Brown Jr., R.M. Fevre M. Geremia R.A. Henrissat B. J. Bacteriol. 1995; 177: 1419-1424Crossref PubMed Google Scholar) predicts that HA synthesis occurs by addition to the reducing end, as suggested by Prehm (41Prehm P. Biochem. J. 1983; 211: 191-198Crossref PubMed Scopus (120) Google Scholar). However, this model is not consistent with recent reports that HA synthesis (42Asplund T. Brinck J. Suzuke M. Briskin J. Heldin P. Biochim. Biophys. Acta. 1998; 1380: 377-388Crossref PubMed Scopus (44) Google Scholar) and Type 3 polysaccharide synthesis, mediated by the related synthase from Streptococcus pneumoniae (43Cartee R.T. Forsee W.T. Yother J.L. Schutzbach J.S. Glycobiology. 1998; 8: 74Google Scholar), occur from the nonreducing end of the growing polysaccharide chains. Definitive answers regarding the direction of polysaccharide chain growth for the HASs and related synthases may require the identification, perhaps utilizing mass spectrometry, of putative UDP-polymer intermediates with the UDP attached at the reducing end.Stoolmiller and Dorfman (44Stoolmiller A.C. Dorfman A. J. Biol. Chem. 1969; 244: 236-246Abstract Full Text PDF PubMed Google Scholar) reported the apparentK UDP-GlcUA andK UDP-GlcNAc as 50 μm and 0.5 mm, respectively, in isolated S. pyogenesmembranes. Their UDP-GlcUA kinetic results gave a linear Lineweaver-Burk plot. They noted, however, that the kinetics of UDP-GlcNAc utilization did not behave in the same linear manner when plotted in double-reciprocal form. These investigators were therefore the first to note sigmoidal behavior of spHAS in streptococcal membranes in response to increasing UDP-GlcNAc concentration. van de Rijn and Drake (34van de Rijn I. Drake R.R. J. Biol. Chem. 1992; 267: 24302-24306Abstract Full Text PDF PubMed Google Scholar) reported, for detergent-solubilized spHAS, values of 39 μm for K UDP-GlcUA and 150 μm for K UDP-GlcNAc, but did not detect a sigmoidal response of the velocity saturation profile when UDP-GlcNAc was varied. They also reported the specific activity of spHAS as 19.4 nmol of UDP-GlcUA/h/mg of extracted membrane protein. We have reported in the accompanying paper (32Tlapak-Simmons V.L. Baggenstoss B.A. Clyne T. Weigel P.H. J. Biol. Chem. 1999; 274: 4239-4245Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) that the specific activities of purified spHAS and seHAS are 5,500 and 12,000 nmol/h/mg, respectively. These values are consistent with spHAS composing only ∼0.3% of the membrane protein in S. pyogenes cells (17Kumari K. Weigel P.H. J. Biol. Chem. 1997; 272: 32539-32546Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The addition of bovine CL substantially increased the specific activity of purified spHAS and seHAS, respectively, to 20,000 and 35,000 nmol/h/mg. The catalytic constants for purified spHAS and seHAS in the presence of bovine CL at 30 °C were, respectively, 22 and 36 monosaccharides/s. These values are in close agreement with those reported for seHAS assayed at 37 °C in whole membranes (17Kumari K. Weigel P.H. J. Biol. Chem. 1997; 272: 32539-32546Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar).The above apparent Michaelis-Menten values for the two substrates were determined using either crude streptococcal membranes or detergent-solubilized membrane extracts, both of which contain other sugar nucleotide-binding proteins and glycosyltransferases and which could contain potential regulatory factors for the HAS enzyme. Here, for the first time, we have characterized the kinetic behavior of purified spHAS and seHAS and these enzymes in membranes containing no other streptococcal proteins. The results demonstrate that then-dodecyl β-d-maltoside-solubilized, purified enzymes behave very similarly to the membrane-bound enzymes. This is an important finding because many studies have reported that HAS activity is irreversibly lost upon solubilization of the protein in a wide variety of nonionic detergents (45Triscott M.X. van de Rijn I. J. Biol. Chem. 1986; 261: 6004-6009Abstract Full Text PDF PubMed Google Scholar). TheK UDP-GlcUA values for membrane-bound spHAS and seHAS were 40 and 51 μm, respectively. TheK UDP-GlcNAc values for membrane-bound spHAS and seHAS were 149 and 60 μm, respectively.Detergent-solubilized, purified HASs showed essentially the same kinetic characteristics as the membrane-bound enzymes, with the exception of their K m values. TheK UDP-GlcUA values increased ∼4-fold for purified spHAS and seHAS, although the latter enzyme was not saturated even at 1.5 mm. Both enzymes also displayed increasedK m values for UDP-GlcNAc after purification; theK UDP-GlcNAc values increased 4-fold for spHAS and 5-fold for seHAS. We also noted that upon storage at −80 °C in the absence of CL, these enzymes slowly lost activity (t12∼2–3 months), and the biggest change appeared to be in K UDP-GlcNAc, which got progressively larger with time of storage. The stimulation of either HAS by CL is due to a large decrease inK UDP-GlcNAc and an increase inV max (32Tlapak-Simmons V.L. Baggenstoss B.A. Clyne T. Weigel P.H. J. Biol. Chem. 1999; 274: 4239-4245Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar).Based on our recent findings, the sensitivity of HASs to detergent solubilization can now be explained. Radiation inactivation analysis revealed that the active spHAS and seHAS species are monomers of the HAS protein in complex with ∼16 CL molecules (46Tlapak-Simmons V.L. Kempner E.S. Baggenstoss B.A. Weigel P.H. J. Biol. Chem. 1998; 273: 26100-26109Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). This conclusion is supported by the results in the accompanying paper (32Tlapak-Simmons V.L. Baggenstoss B.A. Clyne T. Weigel P.H. J. Biol. Chem. 1999; 274: 4239-4245Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) showing that the activity of affinity-purified HAS, which has been depleted of CL, is very low. The HAS enzymes are highly lipid-dependent and are most effectively stimulated by CL. Preliminary mass spectroscopic analysis indicated that even when purified in the absence of exogenous CL, the enzymes still contain residual associated CL. Therefore, the likely reason for why most detergents inactivate HAS (47Ng K.F. Swartz N.B. J. Biol. Chem. 1989; 264: 11776-11783Abstract Full Text PDF PubMed Google Scholar, 48Philipson L.H. Schwartz N.B. J. Biol. Chem. 1984; 259: 5017-5023Abstract Full Text PDF PubMed Google Scholar, 49Prehm P. Mausolf A. Biochem. J. 1986; 235: 887-889Crossref PubMed Scopus (33) Google Scholar) is that these detergents displace the CL required for enzyme activity. Even with CL present, most nonionic detergents will compete more efficiently than CL for interaction with the protein. The mild detergentn-dodecyl β-d-maltoside is apparently strong enough to solubilize HAS from membranes, but not so strong that the enzyme is stripped of CL. Identification of n-dodecyl β-d-maltoside as a useful detergent for solubilizing HAS was a substantial contribution (45Triscott M.X. van de Rijn I. J. Biol. Chem. 1986; 261: 6004-6009Abstract Full Text PDF PubMed Google Scholar).Two substantial differences are apparent between the two enzymes. First, the seHAS enzyme is intrinsically about twice as active as spHAS. This was apparent in this and the accompanying study (32Tlapak-Simmons V.L. Baggenstoss B.A. Clyne T. Weigel P.H. J. Biol. Chem. 1999; 274: 4239-4245Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) with both membrane-bound and purified enzymes and in an earlier study (17Kumari K. Weigel P.H. J. Biol. Chem. 1997; 272: 32539-32546Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) that examined the rates of HA chain elongation by gel filtration analysis. Since the Group C HA capsule is typically larger than the Group A capsule, this difference could be due to theV max between the two HASs. Second, spHAS, but not seHAS, is complexly regulated by UDP-GlcNAc. The spHAS interaction with this substrate shows a cooperative activation of the enzyme as the UDP-GlcNAc concentration increases. This sigmoidal behavior indicates that spHAS has a second binding site for UDP-GlcNAc that is involved in regulation rather than catalysis. Such allosteric-like regulation is usually observed in enzymes that function as oligomers, not enzymes that are active as a monomeric species such as HAS.For a bacterium to synthesize a HA capsule, three different genes must usually be present. These genes, which encode three different enzymes, are arranged in an operon designated the HA synthesis (orhas) operon (50Crater D.L. van de Rijn I. J. Biol. Chem. 1995; 270: 18452-18458Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 51DeAngelis P.L. Weigel P.H. Dev. Biol. Stand. 1995; 85: 225-229PubMed Google Scholar). Two of the enzymes are needed for the cell to produce large amounts of the two UDP-sugar precursors, and the third enzyme is HAS, encoded by the gene hasA. UDP-Glc dehydrogenase (whose gene is designated hasB) is required to make UDP-GlcUA from UDP-Glc in an oxidation reaction that utilizes 2 mol of NAD+/mol of UDP-Glc. UDP-Glc pyrophosphorylase (thehasC gene) creates UDP-Glc from UTP and Glc-1-P. Since UDP-Glc is the precursor from which many of the other sugar nucleotides are made, the amount of UDP-Glc produced by a cell will regulate the total amount of all the cell's sugar nucleotides. Bacteria like Group A Streptococcus that make HA capsules usually have two different genes for this pyrophosphorylase enzyme to increase greatly the total amount of cellular sugar nucleotides and thereby to support synthesis of the large amount of HA in the extracellular capsule.If the bacterial cells did not greatly expand their sugar nucleotide pool, the very active HAS would make HA and deplete the cell of UDP-GlcUA and UDP-GlcNAc. Since the latter is also needed for cell wall synthesis, such depletion would stop the cell from growing. This, in fact, occurs when a streptococcal HAS gene is expressed in another bacterial species that produces the two substrates, but lacks the additional enzyme (hasC) needed to enlarge the sugar nucleotide pools (14DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 14568-14571Abstract Full Text PDF PubMed Google Scholar, 15DeAngelis P.L. Papaconstantinou J. Weigel P.H. J. Biol. Chem. 1993; 268: 19181-19184Abstract Full Text PDF PubMed Google Scholar, 16DeAngelis P.L. Weigel P.H. Biochemistry. 1994; 33: 9033-9039Crossref PubMed Scopus (96) Google Scholar). These cells do not grow well.The cooperative kinetic response of spHAS to UDP-GlcNAc may reflect an evolutionary adaptation by Group A Streptococcus to ensure that cell growth is not impaired by the production of the HA capsule. Because spHAS activity responds to changes in UDP-GlcNAc concentration in a sigmoidal manner, the enzyme cannot attain itsV max until this concentration is very high. This kinetic regulation of spHAS may ensure that cell wall synthesis does not compete with capsule production for UDP-GlcNAc. It is unclear why the seHAS enzyme is not similarly regulated by UDP-GlcNAc or if this lack of regulation is, in fact, a reason why Group A organisms are more pathogenic in humans than Group C strains. Availability of purified streptococcal HASs will facilitate future studies on their role in bacterial virulence and the mechanisms by which these enzymes perform the multiple functions required for HA biosynthesis. HA 1The abbreviations used are: HA, hyaluronan or hyaluronic acid; HAS, hyaluronan synthase; seHAS, S. equisimilis hyaluronan synthase; spHAS, S. pyogeneshyaluronan synthase; CL, cardiolipin.1The abbreviations used are: HA, hyaluronan or hyaluronic acid; HAS, hyaluronan synthase; seHAS, S. equisimilis hyaluronan synthase; spHAS, S. pyogeneshyaluronan synthase; CL, cardiolipin. is a polysaccharide composed of two alternating sugars, β1,3-linked glucuronic acid and β1,4-linked N-acetylglucosamine (1Meyer K. Palmer J.W. J. Biol. Chem. 1934; 107: 629-634Abstract Full Text PDF Google Scholar). Although the structure of HA seems quite simple, the molecule, nonetheless, has unusual physical properties that are important for its numerous biological functions (2Evered D. Whelan J. CIBA Found. Symp. 1989; 143: 1-288Google Scholar, 3Laurent T.C. Acta Oto-laryngol. 1987; 442: 7-24Crossref Scopus (217) Google Scholar, 4Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1242Crossref PubMed Scopus (599) Google Scholar, 5Toole B.P. Hay E.D. Cell Biology of the Extracellular matrix. Plenum Press, New York1991: 305-341Google Scholar). For example, HA forms very viscous solutions and gels due to its high molecular mass and its ability to bind cations and to hydrate large amounts of water. This characteristic of HA provides the viscous lubrication of synovial fluid and helps provide cartilage with its viscoelasticity. These characteristics are also ideal for the role HA has in the extracellular matrices (4Knudson C.B. Knudson W. FASEB J. 1993; 7: 1233-1242Crossref PubMed Scopus (599) Google Scholar, 5Toole B.P. Hay E.D. Cell Biology of the Extracellular matrix. Plenum Press, New York1991: 305-341Google Scholar) of the skin and virtually every vertebrate tissue as well as in the fluid of the vitreous humor of the eye. HA also plays an important role in morphogenesis, wound healing (6Abatangelo G. Martinelli M. Vecchia P. J. Surg. Res. 1983; 35: 410-416Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 7Balazs E.A. Denlinger J.L. J. Rheumatol. 1993; 20: 3-9Google Scholar, 8Goa K.L. Benfield P. Drugs. 1994; 47: 536-566Crossref PubMed Scopus (379) Google Scholar, 9Gressner A.M. Bachem M.G. Digestion. 1995; 56: 335-346Crossref PubMed Scopus (222) Google Scholar), and angiogenesis (10West D.C. Hampson I.N. Arnold F. Kumars S. Science. 1985; 228: 1324-1326Crossref PubMed Scopus (961) Google Scholar, 11Feinberg R.N. Beebe D.C. Science. 1983; 220: 1177-1179Crossref PubMed Scopus (299)
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