Functional Characterization of Enzymatic Steps Involved in Pyruvylation of Bacterial Secondary Cell Wall Polymer Fragments

Abstract

Various mechanisms of protein cell surface display have evolved during bacterial evolution. Several Gram-positive bacteria employ S-layer homology (SLH) domain-mediated sorting of cell-surface proteins and concomitantly engage a pyruvylated secondary cell-wall polymer as a cell-wall ligand. Specifically, pyruvate ketal linked to β-D-ManNAc is regarded as an indispensable epitope in this cell-surface display mechanism. That secondary cell wall polymer (SCWP) pyruvylation and SLH domain-containing proteins are functionally coupled is supported by the presence of an ortholog of the predicted pyruvyltransferase CsaB in bacterial genomes, such as those of Bacillus anthracis and Paenibacillus alvei. The P. alvei SCWP, consisting of pyruvylated disaccharide repeats [→4)-β-D-GlcNAc-(1→3)-4,6-Pyr-β-D-ManNAc-(1→] serves as a model to investigate the widely unexplored pyruvylation reaction. Here, we reconstituted the underlying enzymatic pathway in vitro in combination with synthesized compounds, used mass spectrometry, and nuclear magnetic resonance spectroscopy for product characterization, and found that CsaB-catalyzed pyruvylation of β-D-ManNAc occurs at the stage of the lipid-linked repeat. We produced the P. alvei TagA (PAV_RS07420) and CsaB (PAV_RS07425) enzymes as recombinant, tagged proteins, and using a synthetic 11-phenoxyundecyl-diphosphoryl-α-GlcNAc acceptor, we uncovered that TagA is an inverting UDP-α-D-ManNAc:GlcNAc-lipid carrier transferase, and that CsaB is a pyruvyltransferase, with synthetic UDP-α-D-ManNAc and phosphoenolpyruvate serving as donor substrates. Next, to substitute for the UDP-α-D-ManNAc substrate, the recombinant UDP-GlcNAc-2-epimerase MnaA (PAV_RS07610) of P. alvei was included in this in vitro reconstitution system. When all three enzymes, their substrates and the lipid-linked GlcNAc primer were combined in a one-pot reaction, a lipid-linked SCWP repeat precursor analog was obtained. This work highlights the biochemical basis of SCWP biosynthesis and bacterial pyruvyl transfer.

Pyruvate Substitutions on Glycoconjugates

“…Fifty-four thousand specific hits within the conserved protein domain family SLH (pfam00395), showing up most prevalent in the Firmicutes , Cyanobacteria , and Actinobacteria phyla of bacteria, emphasize the prevalence of this protein domain. Several bacteria synthesizing a suite of SLH proteins contain pyruvate in their cell wall and have a pyruvyltransferase CsaB ortholog [99,109], which indicates a functional coupling of SLH domains and SCWP pyruvylation.…”

“…The most evident difference between these SCWPs is the presence of 4,6Pyr-β- d -ManNAc exclusively at the terminal repeat in the former SCWPs, while in that of P. alvei , the β- d -ManNAc of each repeat is pyruvylated. This might explain the essentiality of the pyruvyltransferase CsaB in the latter organism [109]. It is conceivable to assume that mono- versus poly-pyruvylation of β- d -ManNAc has implications with regard to the biosynthetic pathway of the respective SCWP, especially the mode of activity of the cognate pyruvyltransferase (compare with Section 5.1.1.…”

“…P. alvei possesses a polycistronic SCWP biosynthesis gene cluster comprised of csaB , tagA, tagO , two SLH-protein encoding genes— slhA and spaA —and two ORFs of unknown function [109,122]. For SCWP assembly in P. alvei , distinct enzymatic steps have been investigated [109].…”

“…P. alvei possesses a polycistronic SCWP biosynthesis gene cluster comprised of csaB , tagA, tagO , two SLH-protein encoding genes— slhA and spaA —and two ORFs of unknown function [109,122]. For SCWP assembly in P. alvei , distinct enzymatic steps have been investigated [109]. The bacterium utilizes the subsequent TagO [123] and TagA [124,125] catalysed reactions—typically involved in WTA biosynthesis to produce the undp-PP-bound murein linkage unit [97,126,127] for the biosynthesis of the lipid-linked disaccharide substrate needed to generate the repeat unit backbone of its SCWP [15].…”

The predominance of nucleotidyl activation in bacterial phosphonate biosynthesis

“…Modulating biological function by adding or removing small molecules on cell surface materials has long been recognized as a key mechanism of bacterial adaptation and virulence. Common cell surface modifications include the addition of acetyl 11 , pyruvyl 12 , phosphoethanolamine 13,14 , and phosphocholine (P-Cho) 15–17 groups to lipids and glycans. Indeed, P-Cho is a virulence factor encoded by the Lic pathway in pathogens, such as Streptococcus pneumoniae 15,18 , and lic gene disruption impedes P-Cho decoration of teichoic acids and attenuates S. pneumoniae virulence 19–21 .…”