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Structure-function studies of the C3/C5 epimerases and C4 reductases of the Campylobacter jejuni capsular heptose modification pathways

DOI: 10.1016/j.jbc.2021.100352 DOI Help

Authors: Heba Barnawi (University of Western Ontario) , Laura Woodward (St Andrews University) , Natalie Fava (University of Western Ontario) , Mikhail Roubakha (University of Western Ontario) , Steve D. Shaw (University of Western Ontario) , Chelsea Kubinec (University of Western Ontario) , James H. Naismith (St Andrews University; Rosalind Franklin Institute; University of Oxford) , Carole Creuzenet (University of Western Ontario)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Biological Chemistry , VOL 48

State: Published (Approved)
Published: January 2021

Open Access Open Access

Abstract: Many bacteria produce polysaccharide-based capsules that protect them from environmental insults and play a role in virulence, host invasion, and other functions. Understanding how the polysaccharide components are synthesized could provide new means to combat bacterial infections. We have previously characterized two pairs of homologous enzymes involved in the biosynthesis of capsular sugar precursors GDP-6-deoxy-D-altro-heptose and GDP-6-OMe-L-gluco-heptose in Campylobacter jejuni. However, the substrate specificity and mechanism of action of these enzymes – C3 and/or C5 epimerases DdahB and MlghB and C4 reductases DdahC and MlghC – are unknown. Here, we demonstrate that these enzymes are highly specific for heptose substrates, using mannose substrates inefficiently with the exception of MlghB. We show that DdahB and MlghB feature a jellyroll fold typical of cupins, which possess a range of activities including epimerizations, GDP occupying a similar position as in cupins. DdahC and MlghC contain a Rossman fold, a catalytic triad and a small C-terminal domain typical of short-chain dehydratase reductase enzymes. Integrating structural information with site-directed mutagenesis allowed us to identify features unique to each enzyme and provide mechanistic insight. In the epimerases, mutagenesis of H67, D173, N121, Y134 and Y132 suggested the presence of alternative catalytic residues. We showed that the reductases could reduce GDP-4-keto-6-deoxy-mannulose without prior epimerization though DdahC preferred the pre-epimerized substrate, and identified T110 and H180 as important for substrate specificity and catalytic efficacy. This information can be exploited to identify inhibitors for therapeutic applications or to tailor these enzymes to synthesise novel sugars useful as glycobiology tools.

Journal Keywords: Campylobacter jejuni; capsule; heptose; epimerase; reductase

Subject Areas: Biology and Bio-materials

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

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