Structural and functional evidence for two separate oligosaccharide binding sites of Pasteurella multocida hyaluronan synthase

Abstract

Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing a β1,3-glucuronyltransferase and β1,4-N-acetylglucosaminetransferase domain. PmHAS catalyzes the elongation of hyaluronan (HA) through the sequential addition of single monosaccharides to the non-reducing end of the hyaluronan chain. Research is focused on the relation between the length of the HA oligosaccharide and the single-step elongation kinetics from HA 4 up to HA 9. It was found that the turnover number k cat increased with length to maximum values of 11 and 14 s −1 for NAc-and UA-transfer, respectively. Interestingly, the specificity constant k cat /K M increased with polymer length from HA 5 to HA 7 to a value of 44 mM −1 •s −1 , indicating an oligosaccharide binding site with increasing specificity towards a heptasaccharide at the UA domain. The value of k cat /K M remained moderately constant around 8 mM −1 •s −1 for HA 4 , HA 6 , and HA 8 , indicating a binding site with significantly lower binding specificity at the NAc domain than at the UA domain. These findings are further corroborated by a structural homology model of PmHAS, revealing two distinct sites for binding of oligosaccharides of different sizes, one in each transferase domain. Structural alignment studies between PmHAS and glycosyltransferases of the GT-A fold showed significant similarity in the binding of the UDP-sugars and the orientation of the acceptor substrate. These similarities in substrate orientation in the active site and in essential amino acid residues involved in substrate binding were utilized to localize the two HA oligosaccharide binding sites.

Identification and Comparison of Hyaluronic Acid Biosynthetic Genes from Different Capsular Types of Pasteurella Multocida

“…In contrast, some of the GlmU and HyaD binding pockets were at the catalytic sites. HyaD also contains two active sites: one site has glucuronic acid-transferase activity, which elongates UDPglucuronic acid to oligosaccharides, and the other site has glucosamine-1-P-transferase activity, which prolongs UDP-N-acetyl-D-glucosamine to oligosaccharides [35]. Surface variation in the binding sites of the HyaD protein in this study was consistent with previous studies that identi ed the enzymatic binding site in the glucuronic acid-transferase domain of HyaD [35].…”

“…HyaD also contains two active sites: one site has glucuronic acid-transferase activity, which elongates UDPglucuronic acid to oligosaccharides, and the other site has glucosamine-1-P-transferase activity, which prolongs UDP-N-acetyl-D-glucosamine to oligosaccharides [35]. Surface variation in the binding sites of the HyaD protein in this study was consistent with previous studies that identi ed the enzymatic binding site in the glucuronic acid-transferase domain of HyaD [35]. Our study found that the arrangement of DUF2536 and TPR_2 repeats in the HyaD protein differed in these P. multocida strains, suggesting the reason for the structural variation of the binding site and perhaps different molecular activities.…”

Biotechnological production of hyaluronic acid: a mini review

“…pmHAS has been expressed and successfully synthesised HA in a number of bacterial species, including E. coli (Liu et al 2011 ; Mao and Chen 2007 ; Mao et al 2009 ). Since then this enzyme has become a focal point for cell-free HA production as its truncated form (pmHAS1-703) does not need to be bound to a membrane in order to synthesise HA (DeAngelis et al 1998 ; Jing and DeAngelis 2003 , 2004 ; Kooy et al 2013 ).…”

Directed Evolution of Hyaluronic Acid Synthase from Pasteurella multocida towards High‐Molecular‐Weight Hyaluronic Acid

“…A model of the core pmHAS‐WT 72–688 has already been reported by Kooy and co‐workers . In our model, we completed the C‐terminal portion of the core synthase and improved the N‐terminal domain by further extending it by 40 residues.…”

“…This corroborates that domain A1 confers GlcNAc‐transferase activity . Lastly, pmHAS catalyses the sequential elongation of HA at the nonreducing end of the nascent HA polymer, with its β1,3‐glucuronyltransferase and β1,4‐ N ‐acetylglucosaminyltransferase domains working synergistically as one polypeptide . Due to the processive mechanism of HA polymerisation, it is concluded that, apart from the two UDP sugar binding sites, two other HA binding sites exist in pmHAS .…”