X-ray crystallographic structure of a bacterial polysialyltransferase provides insight into the biosynthesis of capsular polysialic acid

Abstract

Polysialic acid (polySia) is a homopolymeric saccharide that is associated with some neuroinvasive pathogens and is found on selective cell types in their eukaryotic host. The presence of a polySia capsule on these bacterial pathogens helps with resistance to phagocytosis, cationic microbial peptides and bactericidal antibody production. The biosynthesis of bacterial polySia is catalysed by a single polysialyltransferase (PST) transferring sialic acid from a nucleotide-activated donor to a lipid-linked acceptor oligosaccharide. Here we present the X-ray structure of the bacterial PST from Mannheimia haemolytica serotype A2, thereby defining the architecture of this class of enzymes representing the GT38 family. The structure reveals a prominent electropositive groove between the two Rossmann-like domains forming the GT-B fold that is suitable for binding of polySia chain products. Complex structures of PST with a sugar donor analogue and an acceptor mimetic combined with kinetic studies of PST active site mutants provide insight into the principles of substrate binding and catalysis. Our results are the basis for a molecular understanding of polySia biosynthesis in bacteria and might assist the production of polysialylated therapeutic reagents and the development of novel antibiotics.

A New Family of Capsule Polymerases Generates Teichoic Acid-Like Capsule Polymers in Gram-Negative Pathogens

“…The characteristic elements of both folds are two Rossmann-like domains that are either tightly associated, forming a central, continuous β-sheet (GT-A), or opposed to each other, forming a deep cleft that contains the catalytic center (GT-B) ( 11 , 12 ). Polymerases with GT activity are either single-domain enzymes acting alone to synthesize a homopolymer ( 13 – 16 ) or, when generating a heteropolymer, acting in concert with another domain of the same GT fold type, which is either independently expressed ( 1 , 17 ) or part of the same polypeptide ( 18 – 21 ).…”

Processivity in Bacterial Glycosyltransferases

“…56 A comparison of structural data of MhPST and a mammalian PST (ST8SiaIII) revealed that, although both enzymes create the same type of α-2 → 8-linked polySia chains, the enzymes share no common structural features. 57 In Neisseria meningitidis serogroups B (NmB) and C (NmC) and Escherichia coli K1, the polySia in the capsule is essential for virulence, making the polySia-producing enzymes a potential antimicrobial target. The structural differences between the bacterial and mammalians PSTs, leading to higher substrate promiscuity for the first, may aid in selective inhibitor development.…”

Super-Resolution Fluorescence Microscopy Study of the Production of K1 Capsules byEscherichia coli: Evidence for the Differential Distribution of the Capsule at the Poles and the Equator of the Cell

“…Most studies have been focused on the biochemical synthesis of capsular polysaccharides and the molecular machines used to control the biosynthesis pathways. 14 Recent models have concluded that in the case of the E. coli K1 polysaccharide the capsular polysaccharide chains are made inside bacterial cells before transport across the periplasmic space onto the outer membrane. 15 In addition to biosynthesis mechanisms, bacterial capsular morphology has been observed on fixed bacterial surfaces using scanning electron microscopy, transmission electron microscopy, 16,17 and atomic force microscopy.…”

“…Due to the high virulence of bacteria with capsular polysaccharides, the manipulation of polysaccharide production could have important medical benefits. Most studies have been focused on the biochemical synthesis of capsular polysaccharides and the molecular machines used to control the biosynthesis pathways . Recent models have concluded that in the case of the E.…”