Molecular modelling and site-directed mutagenesis provide insight into saccharide pyruvylation by the Paenibacillus alvei CsaB enzyme.

Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic ß-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and KM and kcat values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly.

Assaying Paenibacillus alvei CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release.

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a ß-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium's cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s-1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s-1 were revealed. P. alvei CsaB was inactive on synthetic pNP-ß-d-ManNAc and ß-d-ManNAc-ß-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.

LytR-CpsA-Psr Glycopolymer Transferases: Essential Bricks in Gram-Positive Bacterial Cell Wall Assembly.

The cell walls of Gram-positive bacteria contain a variety of glycopolymers (CWGPs), a significant proportion of which are covalently linked to the peptidoglycan (PGN) scaffolding structure. Prominent CWGPs include wall teichoic acids of Staphylococcus aureus, streptococcal capsules, mycobacterial arabinogalactan, and rhamnose-containing polysaccharides of lactic acid bacteria. CWGPs serve important roles in bacterial cellular functions, morphology, and virulence. Despite evident differences in composition, structure and underlaying biosynthesis pathways, the final ligation step of CWGPs to the PGN backbone involves a conserved class of enzymes-the LytR-CpsA-Psr (LCP) transferases. Typically, the enzymes are present in multiple copies displaying partly functional redundancy and/or preference for a distinct CWGP type. LCP enzymes require a lipid-phosphate-linked glycan precursor substrate and catalyse, with a certain degree of promiscuity, CWGP transfer to PGN of different maturation stages, according to in vitro evidence. The prototype attachment mode is that to the C6-OH of N-acetylmuramic acid residues via installation of a phosphodiester bond. In some cases, attachment proceeds to N-acetylglucosamine residues of PGN-in the case of the Streptococcus agalactiae capsule, even without involvement of a phosphate bond. A novel aspect of LCP enzymes concerns a predicted role in protein glycosylation in Actinomyces oris. Available crystal structures provide further insight into the catalytic mechanism of this biologically important class of enzymes, which are gaining attention as new targets for antibacterial drug discovery to counteract the emergence of multidrug resistant bacteria.

Pyruvate Substitutions on Glycoconjugates.

Glycoconjugates are the most diverse biomolecules of life. Mostly located at the cell surface, they translate into cell-specific "barcodes" and offer a vast repertoire of functions, including support of cellular physiology, lifestyle, and pathogenicity. Functions can be fine-tuned by non-carbohydrate modifications on the constituting monosaccharides. Among these modifications is pyruvylation, which is present either in enol or ketal form. The most commonly best-understood example of pyruvylation is enol-pyruvylation of N-acetylglucosamine, which occurs at an early stage in the biosynthesis of the bacterial cell wall component peptidoglycan. Ketal-pyruvylation, in contrast, is present in diverse classes of glycoconjugates, from bacteria to algae to yeast-but not in humans. Mild purification strategies preventing the loss of the acid-labile ketal-pyruvyl group have led to a collection of elucidated pyruvylated glycan structures. However, knowledge of involved pyruvyltransferases creating a ring structure on various monosaccharides is scarce, mainly due to the lack of knowledge of fingerprint motifs of these enzymes and the unavailability of genome sequences of the organisms undergoing pyruvylation. This review compiles the current information on the widespread but under-investigated ketal-pyruvylation of monosaccharides, starting with different classes of pyruvylated glycoconjugates and associated functions, leading to pyruvyltransferases, their specificity and sequence space, and insight into pyruvate analytics.