Abstract
Three-dimensional structures are not available for polysaccharide synthases and only minimal information on the molecular basis for catalysis is known. The Pasteurella multocida hyaluronan synthase (PmHAS) catalyzes the polymerization of the alternating beta1,3-N-acetylglucosamine-beta1,4-glucuronic acid sugar chain by the sequential addition of single monosaccharides to the non-reducing terminus. Therefore, PmHAS possesses both GlcNAc-transferase and glucuronic acid (GlcUA)-transferase activities. The recombinant Escherichia coli-derived PmHAS enzyme will elongate exogenously supplied hyaluronan chains in vitro with either a single monosaccharide or a long chain depending on the UDP-sugar availability. Competition studies using pairs of acceptors with distinct termini (where one oligosaccharide is a substrate that may be elongated, whereas the other cannot) were performed here; the lack of competition suggests that PmHAS contains at least two distinct acceptor sites. We hypothesize that the size of the acceptor binding pockets of the enzyme corresponds to the size of the smallest high efficiency substrates; thus we tested the relative activity of a series of authentic hyaluronan oligosaccharides and related structural analogs. The GlcUA-transferase site readily elongates (GlcNAc-GlcUA)(2), whereas the GlcNAc-transferase elongates GlcUA-Glc-NAc-GlcUA. The minimally sized oligosaccharides, elongated with high efficiency, both contain a trisaccharide with two glucuronic acid residues that enabled the identification of a synthetic, artificial acceptor for the synthase. PmHAS behaves as a fusion of two complete glycosyltransferases, each containing a donor site and an acceptor site, in one polypeptide. Overall, this information advances the knowledge of glycosaminoglycan biosynthesis as well as assists the creation of various therapeutic sugars for medical applications in the future.
Highlights
Tage of these versatile molecules to produce extracellular glycosaminoglycan capsules, constructing molecular camouflage to avoid host defenses and increase virulence
Analysis of HA Sugars as Acceptors for Pasteurella multocida hyaluronan synthase (PmHAS)—To determine the minimal acceptor structure necessary for efficient HA elongation, we investigated two series of authentic, synthetic hyaluronan ([4GlcUA3GlcNAc]n ϭ [AN]n or [3GlcNAc-4GlcUA]n ϭ [NA]n) oligosaccharides containing a MP group at the reducing termini (Fig. 2)
Disparity among Transferase Site Catalytic Activities—Previous PmHAS investigations demonstrate that two relatively independent active sites on one polypeptide are responsible for the alternating glucuronic acid (GlcUA) and GlcNAc addition to form the HA polymer [2, 3]; molecular genetic disruption of one transferase domain does not affect the catalytic activity of the other domain
Summary
Reagents and Enzyme Preparation—All reagents were of the highest grade available from either Sigma or Fisher, unless otherwise noted. The soluble, truncated dual action PmHAS1–703 enzyme was prepared by chromatography as described previously [10]. The recombinant cells expressing PmHAS1–703 were extracted with 1% (w/v) octyl thioglucoside in 1 M ethylene glycol and 50 mM Hepes, pH 7.2. The clarified extract was purified on a Toyopearl Red AF resin (Tosoh, Montgomeryville, PA) column. The protein was eluted with a NaCl gradient (50 mM Hepes, pH 7.2, 1 M ethylene glycol with 0 –1.5 M NaCl gradient in 1 h). The peak fractions with PmHAS, as assessed by Coomassie Blue staining of SDS-polyacrylamide gels, were pooled and concentrated by ultra-
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