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

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 APIdeficient 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 M. These results support the assertion that Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMPKdo. 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.

Structural analysis of arabinose-5-phosphate isomerase fromBacteroides fragilisand functional implications

“…Interestingly, CMP-Kdo is neither the substrate nor the product of the reaction catalyzed by API, but corresponds to the end product of the Kdo biosynthetic pathway; it could therefore act as a feedback inhibitor. Our conclusions are supported by a recent study that demonstrates that bfAPI has arabinose-5-phosphate isomerase activity and is inhibited by CMP-Kdo (Cech et al, 2014). Here, we report the structural analysis of bfAPI and its functional implications.…”

“…The gutQ gene, on the other hand, is located in an operon involved in d-glucitol (sorbitol) metabolism. However, the involvement of GutQ in the metabolism of d-glucitol, or a link between d-glucitol metabolism and LPS biosynthesis, has not been established (Meredith & Woodard, 2005;Cech et al, 2014). The biological significance of the API redundancy in E. coli is currently unknown, but it is speculated that they may have additional roles besides producing A5P.…”

Identification of a d -Arabinose-5-Phosphate Isomerase in the Gram-Positive Clostridium tetani

“…Mixtures containing 100 mM bis-Tris propane buffer, pH 6.75, 1 mM EDTA, 10% D 2 O, 500 nM CtAPI, and either 5 mM A5P or 5 mM Ru5P were incubated at room temperature (21°C) for 48 h, which was sufficient time to reach equilibrium. The equilibrium constant (K eq ) was determined using 31 P nuclear magnetic resonance (NMR) as previously described (3,23). NMR experiments were performed using a Varian 400 multinuclear NMR spectrometer.…”

“…The initiated reaction mixtures were incubated for 3 min at 37°C and then quenched with an equal volume of 10% (wt/vol) trichloroacetic acid. Color was developed as previously described (3,25). Absorbance was read at 549 nm, and the data were fit using the nonlinear least-squares regression function of GraphPad Prism 5 software.…”

“…The two-domain APIs of Escherichia coli have been classified as L-API (involved in lipopolysaccharide [LPS] biosynthesis), G-API (part of the glucitol phosphotransferase operon), and K-API (in-volved in K-antigen biosynthesis). L-APIs, such as KdsD from E. coli, are the first enzymes in the pathway responsible for the biosynthesis of 3-deoxy-D-manno-octulosonic acid (Kdo), an essential component of both the LPS of Gram-negative bacteria and the rhamnogalacturonan-II of certain plants (1)(2)(3). While still not fully understood, G-APIs, such as GutQ, appear to function in the biosynthesis of A5P to modulate expression levels of the gut operon (4).…”