Processivity in Bacterial Glycosyltransferases

Abstract

Extracellular polysaccharides and glycoproteins of pathogenic bacteria assist in adherence, autoaggregation, biofilm formation, and host immune system evasion. As a result, considerable research in the field of glycobiology is dedicated to study the composition and function of glycans associated with virulence, as well as the enzymes involved in their biosynthesis with the aim to identify novel antibiotic targets. Especially, insights into the enzyme mechanism, substrate binding, and transition-state structures are valuable as a starting point for rational inhibitor design. An intriguing aspect of enzymes that generate or process polysaccharides and glycoproteins is the level of processivity. The existence of enzymatic processivity reflects the need for regulation of the final glycan/glycoprotein length and structure, depending on the role they perform. In this Review, we describe the currently reported examples of various processive enzymes involved in polymerization and transfer of sugar moieties, predominantly in bacterial pathogens, with a focus on the biochemical methods, to showcase the importance of studying processivity for understanding the mechanism.

An enzyme-based protocol for cell-free synthesis of nature-identical capsular oligosaccharides from Actinobacillus pleuropneumoniae serotype 1

“…Previous studies demonstrated that the truncation of the TPR domain changed the oligomerization state of Cps1B from dimeric to monomeric (17). Oligomerization was described as structural basis for the processivity of various classes of enzymes (16,17). In contrast, extended polymer binding sites were shown to mediate processivity in carbohydrate polymerases (14)(15)(16)34).…”

“…Oligomerization was described as structural basis for the processivity of various classes of enzymes (16,17). In contrast, extended polymer binding sites were shown to mediate processivity in carbohydrate polymerases (14)(15)(16)34). Although this study was focused on the biotechnological use of Cps1B, we performed initial limited proteolysis and surface plasmon resonance experiments to clarify if the TPR domain is involved in the binding of non-O-acetylated App1 polymer (data not shown).…”

“…Next, we used the oligosaccharides of pool I to analyze the product profiles generated by Cps1B at varying donor to acceptor (d/a) ratios. Since previous studies have shown that the truncation of the TPR domain influences the oligomerization state of Cps1B (17), and since oligomerization can be a prerequisite for processivity (16), we included Cps1B-ΔTPR in the study. In overnight reactions, Cps1B and Cps1B-ΔTPR were incubated with donor substrates (UDP-Gal and UDP-GlcNAc) and varying oligosaccharide acceptor (pool I) concentrations (d/a 10:1-1,000:1).…”

“…Of special biotechnological value in this context are polymerases deploying a distributive mode of chain elongation, because they allow the generation of product pools of uniform chain length (Gaussian shaped distribution) as well as control over the average degree of polymerization (DP) of the products. In contrast to processive enzymes, which catalyze multiple transfers without releasing the growing polymer (and thus produce a heterogeneous product profile), strictly distributive polymerases transfer only a single sugar moiety at a time and dissociate afterwards, allowing the stoichiometry between donor and acceptor substrate to dictate chain length (14)(15)(16). We recently described the recombinant production of Cps1B, the capsule polymerase of App serotype 1 (17).…”

In vitro biosynthesis of poly-β-1,4-glucan derivatives using a pro-miscuous glycosyltransferase

“…The enzymatic synthesis of cellulose in vitro is challenging because the natural biosynthetic machinery consists of complex membrane-embedded multi component systems 26 , that are hard to express in functional form. Furthermore, biosynthesis is processive and is biased towards production of very long oligomers 27,28 . Recent advances in employing biocatalysts for in vitro cellulose synthesis include production of high molecular weight (DP >200) cellulose or cellulose microfibrils from uridine diphosphate glucose (UDP-Glc) via exploitation of bacterial or plant-derived cellulose synthase (complexes) that are functionally reconstituted in lipid bilayers 29,30 .…”