A Hyaluronidase Supercatalyst for the Enzymatic Polymerization to Synthesize Glycosaminoglycans

Kobayashi, Shiro; Ohmae, Masashi; Ochiai, Hideo; Fujikawa, Shun‐ichi

doi:10.1002/chem.200600191

Cited by 41 publications

(25 citation statements)

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“…The only other reported methods for chemoenzymatically constructing chimeric GAGs involved the use of testicular hyaluronidase to (a) transglycosylate GAG chains (28,29) or (b) couple oxazoline monomers (30). Testicular hyaluronidase catalyzes the cleavage of HA, chondroitin, or chondroitin sulfate using a water hydroxyl with an optimum at pH 5.…”

Section: Resultsmentioning

confidence: 99%

Acceptor Specificity of the Pasteurella Hyaluronan and Chondroitin Synthases and Production of Chimeric Glycosaminoglycans

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Linhardt

et al. 2007

Journal of Biological Chemistry

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The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (ϳ10 2-4 sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes. Glycosaminoglycans (GAGs),2 polysaccharides containing a hexosamine as part of their repeat unit, serve essential biological functions in vertebrates, including adhesion, modulation of motility and proliferation, and coagulation (1-8). Certain pathogenic bacteria camouflage themselves with GAGs to increase virulence and enhance infection in their animal hosts. These capsular polysaccharides serve as molecular camouflage as well as a means to potentially hijack host functions (9). The backbones of the vertebrate GAGs, HA (-␤1,4-GlcUA-␤1,3-GlcNAc-), chondroitin sulfate (-␤1,4-GlcUA-␤1,3-GalNAc-), heparan sulfate (␣1,4-GlcUA-␤1,4-GlcNAc-), heparin (-␣1,4-IdoUA-␣ 1,4-GlcNAc-), and keratan sulfate (-␤1,3-Gal-␤1,4-GlcNAc-), are sulfated except for HA. The bacterial GAG polymers are not known to be sulfated. The enzymes involved in the biosynthesis of all the GAG backbones, except for keratan, have been molecularly cloned.Certain bacterial enzymes, including the Gram-negative Pasteurella multocida type A hyaluronan synthase, PmHAS, and the type F chondroitin synthase, PmCS, are particularly amenable to study because of their two active center architecture (10, 11), their ability to polymerize long chains in vitro (12), and their ability to elongate exogenously supplied acceptor oligosaccharides in vitro (13,14). The Escherichia ...

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Section: Resultsmentioning

confidence: 99%

Acceptor Specificity of the Pasteurella Hyaluronan and Chondroitin Synthases and Production of Chimeric Glycosaminoglycans

Tracy

Avci

Linhardt

et al. 2007

Journal of Biological Chemistry

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“…Enzymatic catalysts can be regarded as a third stream, despite still being small in importance compared with the other two. [25] Enzymatic polymerization is defined as ''the in vitro polymerization of artificial substrate monomers catalyzed by an isolated enzyme via nonbiosynthetic (nonmetabolic) pathways''. [7,9,[12][13][14]17] Enzymatic catalysis involves several characteristics in general: i) high catalytic activity (high turn-over number), ii) reactions under mild conditions with respect to temperature, pressure, solvent, neutral pH, etc, bringing about high energetic efficiency, and, iii) high reaction control of enantio-, chemo-, regio-, stereo-and choroselectivities, giving rise to perfectly structure-regulated products in most cases.…”

Section: Introductionmentioning

confidence: 99%

“…In fact, when a substrate monomer is appropriately designed and combined with a correctly selected enzyme by considering its function, many polymers have been synthesized in the two decades, including polysaccharides, [16,20,25] polyesters, [12,17,21,26] polyaromatics [27] and vinyl polymers. [28] Some of these polymers had not been successfully synthesized via conventional polymerization; that is, enzymatic polymerization enabled their in vitro synthesis for the first time, as exemplified by cellulose, [29] chitin, [30] xylan, [31] hyaluronan, [32] chondrotin, [33] unnatural hybrid polysaccharides, [34] and polyphenol and its derivatives.…”

Section: Introductionmentioning

confidence: 99%

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Recent Developments in Lipase‐Catalyzed Synthesis of Polyesters

Kobayashi

2009

Macromol. Rapid Commun.

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254

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Polyester synthesis by lipase catalyst involves two major polymerization modes: i) ring-opening polymerization of lactones, and, ii) polycondensation. Ring-opening polymerization includes the finding of lipase catalyst; scope of reactions; polymerization mechanism; ring-opening polymerization reactivity of lactones; enantio-, chemo- and regio-selective polymerizations; and, chemoenzymatic polymerizations. Polycondensation includes polymerizations involving condensation reactions between carboxylic acid and alcohol functional groups to form an ester bond. In most cases, a carboxylic acid group is activated as an ester form, such as a vinyl ester. Many recently developed polymerizations demonstrate lipase catalysis specific to enzymatic polymerization and appear very useful. Also, since lipase-catalyzed polyester synthesis provides a good opportunity for conducting "green polymer chemistry", the importance of this is described.

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“…Furthermore Kobayashi et al were able to show that hyaluronidase is able to catalyze the in vitro synthesis of hyaluronan [228] , chondroitin [229] , chondroitin sulfate [230] , their derivatives [229,231] and of non -natural GAGs [232] .…”

Section: Glycosidasesmentioning

confidence: 99%