Identification and characterization of a chondroitin synthase from Avibacterium paragallinarum

Abstract

Avibacterium paragallinarum is a Gram-negative bacterium that causes infectious coryza in chicken. It was reported that the capsule polysaccharides extracted from Av. paragallinarum genotype A contained chondroitin. Chondroitin synthase of Av. paragallinarum (ApCS) encoded by one gene within the presumed capsule biosynthesis gene cluster exhibited considerable homology to identified bacterial chondroitin synthases. Herein, we report the identification and characterization of ApCS. This enzyme indeed displays chondroitin synthase activity involved in the biosynthesis of the capsule. ApCS is a bifunctional protein catalyzing the elongation of the chondroitin chain by alternatively transferring the glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) residues from their nucleotide forms to the non-reducing ends of the saccharide chains. GlcA with a para-nitrophenyl group (pNP) could serve as the acceptor for ApCS; this enzyme shows a stringent donor tolerance when the acceptor is as small as this monosaccharide. Then, UDP-GalNAc and GlcA-pNP were injected sequentially through the chip-immobilized chondroitin synthases, and the surface plasmon resonance data demonstrated that the up-regulated extent caused by the binding of the donor is one possibly essential factor in successful polymerization reaction. This conclusion will, therefore, enhance the understanding of the mode of action of glycosyltransferase. Surprisingly, high activity at near-zero temperature as well as weak temperature dependence of this novel bacterial chondroitin synthase indicate that ApCS was a cold-active enzyme. From all accounts, ApCS becomes the fourth known bacterial chondroitin synthase, and the potential applications in artificial chondroitin sulfate and glycosaminoglycan synthetic approaches make it an attractive glycosyltransferase for further investigation.

Heparosan oligosaccharide synthesis using engineered single-function glycosyltransferases

“…Interestingly, one chondroitin synthase (EC 2.4.1.175) has been identified in Genotype I strains of A. paragallinarum . 31 Furthermore, three-dimensional protein structure models of these putative KfiAs were respectively built based on the structure of EcKfiA (PDB code , https://www.rcsb.org/structure/). The significant amino acid sequence homology between these putative KfiAs and EcKfiA encouraged us to characterize these proteins (Fig.…”

“…UDP- N -acetylglucosamine (UDP-GlcNAc) was also purchased from Sigma. UDP-GlcNTFA, 39 UDP-GlcNAz, 31,32,40 and UDP-GlcA 41 were synthesized following a protocol described previously.…”

“…PAMN-HPLC and ESI-MS were used to determine the reaction products, as previously described. 31 The structure of the final products was confirmed by NMR using protocols described previously. 43…”

Cascade synthesis of uridine-5′-diphosphate glucuronic acid by coupling multiple whole cells expressing hyperthermophilic enzymes

“…PAMN-HPLC analysis was performed using a Shimadzu HPLC instrument (Tokyo, Japan) equipped with a YMC-Pack Polyamine II column (250 × 4.6 mm; Shimogyo-ku, Kyoto, Japan) [14]. The column was eluted at a flow rate of 0.5 mL/min, with a linear gradient of KH 2 PO 4 from 0 to 0.6 M over 40 min for UDP-Glc detection, and a linear gradient of KH 2 PO 4 from 0.2 to 0.6 M over 40 min for UDP-GlcA detection.…”

Heterologous production of chondroitin

“…Biosynthesis of HP from microbial sources requires the microbial production of the precursor heparosan [ 178 , 179 , 180 ] with further chemical and/or enzymatic modifications [ 19 , 181 , 182 , 183 , 184 ]. E. coli K5, P. multocida type D and Avibacterium paragallinarum genotype II can synthesize heparosan [ 35 , 36 , 185 , 186 ]. High titers of heparosan have been achieved using E. coli K5 (up to 15 g/L in bioreactor) [ 178 , 179 ] which has the heparosan biosynthetic genes encoding glycosyltransferases and dehydrogenases organized in an operon ( kfiA-D, Fig.…”