PseG of Pseudaminic Acid Biosynthesis

Abstract

The flagellin proteins in pathogenic bacteria such as Campylobacter jejuni and Helicobacter pylori are heavily glycosylated with the nine-carbon ␣-keto acid, pseudaminic acid. The presence of this posttranslational modification is absolutely required for assembly of functional flagella. Since motility is required for colonization, pseudaminic acid biosynthesis represents a virulence factor in these bacteria. Pseudaminic acid is generated from UDP-N-acetylglucosamine in five biosynthetic steps. The final step has been shown to involve the condensation of 2,4-diacetamido-2,4,6-trideoxy-L-altrose (6-deoxy-AltdiNAc) with phosphoenolpyruvate as catalyzed by the enzyme pseudaminic acid synthase, NeuB3. The 6-deoxy-AltdiNAc used in this process is generated from its nucleotide-linked form, UDP-6-deoxy-AltdiNAc, by the action of a hydrolase that cleaves the glycosidic bond and releases UDP. This manuscript describes the first characterization of a UDP-6-deoxy-AltdiNAc hydrolase, namely PseG (Cj1312) from C. jejuni. The activity of this enzyme is independent of the presence of divalent metal ions, and the values of the catalytic constants were found to be k cat ‫؍‬ 27 s ؊1 and K m ‫؍‬ 174 M. The enzyme was shown to hydrolyze the substrate with an overall inversion of stereochemistry at C-1 and to utilize a C-O bond cleavage mechanism during catalysis. These results, coupled with homology comparisons, suggest that the closest ancestors to the hydrolase are members of the metal-independent GT-B family of glycosyltransferases that include the enzyme MurG.

Characterization of the emerging zoonotic pathogen Arcobacter thereius by whole genome sequencing and comparative genomics

“…jejuni [78]. Interestingly, this gene region contained also an acetylase (AA347_00585), as well as a gene with high similarity to C .…”

“…This pathway was only partially present in A . butzleri RM4018, as both the pseG (encoding for an hydrolase with important function during the fourth step of the pseudoaminic acid biosynthesis pathway [78]) and pseH genes were missing, suggesting that this post-translationally modification system through O-linked glycosylation is not functional in A . butzleri [79].…”

Structural and functional characterization of the <em>Helicobacter pylori </em>cytidine 5&#39;-monophosphate-pseudaminic acid synthase PseF: molecular insight into substrate recognition and catalysis mechanism

“…Figure 12 shows PPI partner proteins of HpPseF derived from the STRING database. The functional partners are mainly flagella glycosylation pathway proteins (PseB, PseC, PseH, HpPseG, PseI)19,49 and flagellar basal body L-ring protein (FlgH). In addition, HpPseF interacts with other proteins including polysialic acid capsule expression protein (KpsF), 50S ribosomal protein L34 (RpmH) and three hypothetical proteins (HP0465; HP0114, HP1570).…”

Structural Insights into the Glycosyltransferase Activity of the Actinobacillus pleuropneumoniae HMW1C-like Protein

“…Glycosyltransferase Assay of ApHMW1C and Data AnalysisGlycosyltransferase activity of ApHMW1C was assessed as previously described (19,29). Experimental details are given in the supplemental information.…”