Kinetic and structural analysis of two transferase domains inPasteurella multocida hyaluronan synthase

F.K. Kooy, H.H. Beeftink, M.H.M. Eppink, J. Tramper, G. Eggink, C.G. Boeriu

Research output: Contribution to journalArticleAcademicpeer-review

4 Citations (Scopus)

Abstract

Pasteurella multocida hyaluronan synthase (PmHAS) encompasses two transferase domains that elongatea growing hyaluronan (HA) oligosaccharide chain by addition of either GlcNAc or GlcUA residues froma corresponding UDP-sugar. Initial velocity studies of single-step elongations were conducted for bothdomains by independently varying the concentrations of the HA oligosaccharide and the UDP-sugar.Two-substrate models were discriminated by their goodness-of-fit parameters and by dead-end inhi-bition studies. A mechanistic shift from a steady-state ordered bi-bi to rapid equilibrium ordered bi-bimechanism was observed at the NAc-site between the HA6and HA8elongation. This shift was invokedby a minor reduction in turnover number kcat. Both NAc- and UA-transferase domains follow a sequentialkinetic mechanism, most likely an ordered one in which the UDP-sugar donor binds first, followed bythe HA oligosaccharide. After transfer of the sugar moiety, both products are released, first the elongatedHA oligosaccharide and then the UDP sugar. This mechanism was visualized with a structural model ofPmHAS that presented two flexible loops, one in each transferase domain; these loops form a bridgeabove the active site.
Original languageEnglish
Pages (from-to)138-145
Number of pages8
JournalJournal of Molecular Catalysis. B, Enzymatic
Volume102
DOIs
Publication statusPublished - 2014

Keywords

  • blood-group-b
  • enzymological characterization
  • conformational-changes
  • n-acetylglucosamine
  • crystal-structure
  • group-a
  • glycosyltransferase
  • polypeptide
  • mechanism
  • substrate

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