Kinetic and structural analysis of two transferase domains in Pasteurella multocida hyaluronan synthase

Pasteurella multocida hyaluronan synthase (PmHAS) encompasses two transferase domains that elongate a growing hyaluronan (HA) oligosaccharide chain by addition of either GlcNAc or GlcUA residues from a corresponding UDP-sugar. Initial velocity studies of single-step elongations were conducted for both domains 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 inhibition studies. A mechanistic shift from a steady-state ordered bi-bi to rapid equilibrium ordered bi-bi mechanism was observed at the NAc-site between the HA6 and HA8 elongation. This shift was invoked by a minor reduction in turnover number kcat. Both NAc- and UA-transferase domains follow a sequential kinetic mechanism, most likely an ordered one in which the UDP-sugar donor binds first, followed by the HA oligosaccharide. After transfer of the sugar moiety, both products are released, first the elongated HA oligosaccharide and then the UDP sugar. This mechanism was visualized with a structural model of PmHAS that presented two flexible loops, one in each transferase domain; these loops form a bridge above the active site. Based on structural similarities between PmHAS with α1,3-galactosyltransferase (α3GT) and β1,4-galactosyltransferase (β4Gal-T1), these flexible loops are seemingly involved in a conformational change upon binding the UDP-sugar, inducing the ordered mechanism. Kinetic analyses have demonstrated significant differences in dissociation constant KD,UA and KM,NAc of 124 ± 31 μM and 28 ± 8.8 mM, respectively, suggesting that the variance in the binding affinity of the two UDP-sugars controls the molecular weight of hyaluronan.