Structural requirements of d-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Pseudomonas aeruginosa were analysed in detail using advanced NMR techniques. We performed epitope mapping studies of the binding between the enzyme and the most potent KdsD inhibitors found to date, together with studies of a set of newly synthesised arabinose 5-phosphate (A5P) mimetics. We report here the first experimental evidence that KdsD may bind the furanose form of A5P, suggesting that catalysis of ring opening may be an important part of KdsD catalysis.

d-Arabinose 5-phosphate isomerases (APIs) catalyze the interconversion of d-ribulose 5-phosphate and d-arabinose 5-phosphate (A5P). A5P is an intermediate in the biosynthesis of 3-deoxy-d-manno-octulosonate (Kdo), an essential component of lipopolysaccharide, the lipopolysaccharide found in the outer membrane of Gram-negative bacteria. The genome of the Gram-positive pathogen Listeria monocytogenes contains a gene encoding a putative sugar isomerase domain API, Q723E8, with significant similarity to c3406, the only one of four APIs from Escherichia coli CFT073 that lacks a cystathionine-β-synthase domain. However, L. monocytogenes lacks genes encoding any of the other enzymes of the Kdo biosynthesis pathway. Realizing that the discovery of an API in a Gram-positive bacterium could provide insight into an alternate physiological role of A5P in the cell, we prepared and purified recombinant Q723E8. We found that Q723E8 does not possess API activity, but instead is a novel GPI (d-glucose 6-phosphate isomerase). However, the GPI activity of Q723E8 is weak compared with previously described GPIs. L. monocytogenes contains an ortholog of the well-studied two-domain bacterial GPI, so this maybe redundant. Based on this evidence glucose utilization is likely not the primary physiological role of Q723E8.

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PSAp) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PSAp would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PSAp in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PSAp is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PSAp follows Michaelis–Menten kinetics at 60 °C, with a KM for erythrose 4-phosphate of 280 μM, a KM for phosphoenolpyruvate of 891 μM, and a kcat of 1.0 s−1. None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PSAp. The structure of DAH7PSAp is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.Graphical abstractDownload full-size imageHighlights► We investigate the DAH7P synthase from Aeropyrum pernix, a primitive archaebacterium. ► We present the biochemical characterization and crystal structure of the enzyme. ► The enzyme activity is not regulated by metabolites from the shikimate pathway.

This article describes the adaptation of a simple colorimetric assay for inorganic pyrophosphate to the enzyme 3-deoxy-d-manno-octulosonate cytidylyltransferase (CMP–KDO synthetase, KdsB, EC 2.7.7.38), a key enzyme in the biosynthesis of lipopolysaccharide (LPS) in Gram-negative organisms. This assay is particularly useful because it can be combined with the malachite green (MG) assay for inorganic phosphate to form an assay system capable of determining inorganic phosphate and inorganic pyrophosphate in the same solution (the MG/EK (eikonogen reagent) assay). This assay system has the potential for simultaneous screening of the 3-deoxy-d-manno-octulosonate (KDO) biosynthesis pathway. We tested this potential using two enzymes, KdsB and KdsC, involved in the biosynthesis and use of the key bacterial 8-carbon sugar, KDO.

We explored the d-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Francisella tularensis, a highly infectious Gram-negative pathogen that has raised concern as a potential bioweapon, as a target for the development of novel chemotherapeutics. F. tularensis KdsD was expressed in Escherichia coli from a synthetic gene, purified, and characterized. A group of hydroxamates designed to be mimics of the putative enediol intermediate in the enzyme’s catalytic mechanism were prepared and tested as inhibitors of F. tularensis KdsD. The best inhibitor, which has an IC50 of 7 μM, is the most potent KdsD inhibitor reported to date.

The proposed cyclic mechanism of 3-deoxy-d-manno-octulosonic acid 8-phosphate synthase and the mechanism of chorismate mutase share certain structural and electronic similarities. In this report, we examine several inhibitors of chorismate mutase for their efficacy against KDO 8-P synthase.Graphic