Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

Abstract

ABSTRACT Arabinose-5-phosphate isomerases (APIs) catalyze the interconversion of d -ribulose-5-phosphate and d -arabinose-5-phosphate, the first step in the biosynthesis of 3-deoxy- d -manno-octulosonic acid (Kdo), an essential component of the lipopolysaccharide in Gram-negative bacteria. Classical APIs, such as Escherichia coli KdsD, contain a sugar isomerase domain and a tandem cystathionine beta-synthase domain. Despite substantial effort, little is known about structure-function relationships in these APIs. We recently reported an API containing only a sugar isomerase domain. This protein, c3406 from E. coli CFT073, has no known physiological function. In this study, we investigated a putative single-domain API from the anaerobic Gram-negative bacterium Bacteroides fragilis . This putative API (UniProt ID Q5LIW1 ) is the only protein encoded by the B. fragilis genome with significant identity to any known API, suggesting that it is responsible for lipopolysaccharide biosynthesis in B. fragilis . We tested this hypothesis by preparing recombinant Q5LIW1 protein (here referred to by the UniProt ID Q5LIW1 ), characterizing its API activity in vitro , and demonstrating that the gene encoding Q5LIW1 (GenBank ID YP_209877.1 ) was able to complement an API-deficient E. coli strain. We demonstrated that Q5LIW1 is inhibited by cytidine 5′-monophospho-3-deoxy- d - manno -2-octulosonic acid, the final product of the Kdo biosynthesis pathway, with a K i of 1.91 μM. These results support the assertion that Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMP-Kdo. The sugar isomerase domain of E. coli KdsD, lacking the two cystathionine beta-synthase domains, demonstrated API activity and was further characterized. These results suggest that Q5LIW1 may be a suitable system to study API structure-function relationships.