Dataset Information

Biosynthesis of GDP-d-glycero-?-d-manno-heptose for the Capsular Polysaccharide of Campylobacter jejuni.

ABSTRACT: The capsular polysaccharide (CPS) structure of Campylobacter jejuni contributes to its robust fitness. Many strains contain heptose moieties in their CPS units. The precursor heptose is GDP-d-glycero-?-d-manno-heptose; modifications to the stereochemistry at C3-C6 as well as additions of methyl and phosphoramidate groups lend to the hypervariability of the C. jejuni CPS structures. Synthesis of GDP-d-glycero-?-d-manno-heptose has been described previously, but using enzymes from Aneurinibacillus thermoaerophilus DSM 10155. Here we describe the complete synthesis of GDP-d-glycero-?-d-manno-heptose using enzymes from C. jejuni NTCC 11168: Cj1152 and Cj1423-Cj1425. Our results yield kinetic parameters for these enzymes and outline a successful strategy for milligram-gram scale synthesis of GDP-d-glycero-?-d-manno-heptose. This achievement is critical for the characterization of other carbohydrate tailoring enzymes, which are expected to utilize GDP-d-glycero-?-d-manno-heptose for the biosynthesis of more complex carbohydrates in the CPS of C. jejuni.

SUBMITTER: Huddleston JP

PROVIDER: S-EPMC6859792 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Publications

Biosynthesis of GDP-d-glycero-α-d-manno-heptose for the Capsular Polysaccharide of Campylobacter jejuni.

Huddleston Jamison P JP Raushel Frank M FM

Biochemistry 20190829 37

The capsular polysaccharide (CPS) structure of Campylobacter jejuni contributes to its robust fitness. Many strains contain heptose moieties in their CPS units. The precursor heptose is GDP-d-glycero-α-d-manno-heptose; modifications to the stereochemistry at C3-C6 as well as additions of methyl and phosphoramidate groups lend to the hypervariability of the C. jejuni CPS structures. Synthesis of GDP-d-glycero-α-d-manno-heptose has been described previousl ...[more]

PMID: 31449400

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Project description:D-Glycero-d-manno-heptose-1,7-bisphosphate phosphatase (GmhB) is a member of the histidinol-phosphate phosphatase (HisB) subfamily of the haloalkanoic acid dehalogenase (HAD) enzyme superfamily. GmhB supports two divergent biochemical pathways in bacteria: the d-glycero-d-manno-heptose-1alpha-GDP pathway (in S-layer glycoprotein biosynthesis) and the l-glycero-d-manno-heptose-1beta-ADP pathway (in lipid A biosynthesis). Herein, we report the comparative analysis of substrate recognition in selected GmhB orthologs. The substrate specificity of the l-glycero-d-manno-heptose-1beta-ADP pathway GmhB from Escherichia coli K-12 was evaluated using hexose and heptose bisphosphates, histidinol phosphate, and common organophosphate metabolites. Only d-glycero-d-manno-heptose 1beta,7-bisphosphate (k(cat)/K(m) = 7 x 10(6) M(-1) s(-1)) and d-glycero-d-manno-heptose 1alpha,7-bisphosphate (k(cat)/K(m) = 7 x 10(4) M(-1) s(-1)) displayed physiologically significant substrate activity. (31)P NMR analysis demonstrated that E. coli GmhB selectively removes the C(7) phosphate. Steady-state kinetic inhibition studies showed that d-glycero-d-manno-heptose 1beta-phosphate (K(is) = 60 microM, and K(ii) = 150 microM) and histidinol phosphate (K(is) = 1 mM, and K(ii) = 6 mM), while not hydrolyzed, do in fact bind to E. coli GmhB, which leads to the conclusion that nonproductive binding contributes to substrate discrimination. High catalytic efficiency and a narrow substrate range are characteristic of a well-evolved metabolic enzyme, and as such, E. coli GmhB is set apart from most HAD phosphatases (which are typically inefficient and promiscuous). The specialization of the biochemical function of GmhB was examined by measuring the kinetic constants for hydrolysis of the alpha- and beta-anomers of d-glycero-d-manno-heptose 1beta,7-bisphosphate catalyzed by the GmhB orthologs of the l-glycero-d-manno-heptose 1beta-ADP pathways operative in Bordetella bronchiseptica and Mesorhizobium loti and by the GmhB of the d-glycero-d-manno-heptose 1alpha-GDP pathway operative in Bacteroides thetaiotaomicron. The results show that although each of these representatives possesses physiologically significant catalytic activity toward both anomers, each displays substantial anomeric specificity. Like E. coli GmhB, B. bronchiseptica GmhB and M. loti GmhB prefer the beta-anomer, whereas B. thetaiotaomicron GmhB is selective for the alpha-anomer. By determining the anomeric configuration of the physiological substrate (d-glycero-d-manno-heptose 1,7-bisphosphate) for each of the four GmhB orthologs, we discovered that the anomeric specificity of GmhB correlates with that of the pathway kinase. The conclusion drawn from this finding is that the evolution of the ancestor to GmhB in the HisB subfamily provided for specialization toward two distinct biochemical functions.

Dataset Information

Biosynthesis of GDP-d-glycero-?-d-manno-heptose for the Capsular Polysaccharide of Campylobacter jejuni.

Publications

Biosynthesis of GDP-d-<i>glycero</i>-α-d-<i>manno</i>-heptose for the Capsular Polysaccharide of <i>Campylobacter jejuni</i>.

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