Epoxide hydrolases are essential for the processing of epoxide‐containing compounds in detoxification or metabolism. The classic epoxide hydrolases have an α/β hydrolase fold and act via a two‐step reaction mechanism including an enzyme–substrate intermediate. We report here the structure of the limonene‐1,2‐epoxide hydrolase from Rhodococcus erythropolis , solved using single‐wavelength anomalous dispersion from a selenomethionine‐substituted protein and refined at 1.2 Å resolution. This enzyme represents a completely different structure and a novel one‐step mechanism. The fold features a highly curved six‐stranded mixed β‐sheet, with four α‐helices packed onto it to create a deep pocket. Although most residues lining this pocket are hydrophobic, a cluster of polar groups, including an Asp–Arg–Asp triad, interact at its deepest point. Site‐directed mutagenesis supports the conclusion that this is the active site. Further, a 1.7 Å resolution structure shows the inhibitor valpromide bound at this position, with its polar atoms interacting directly with the residues of the triad. We suggest that several bacterial proteins of currently unknown function will share this structure and, in some cases, catalytic properties.

Glutamine synthetase catalyzes the ligation of glutamate and ammonia to form glutamine, with the resulting hydrolysis of ATP. The enzyme is a central component of bacterial nitrogen metabolism and is a potential drug target. Here, we report a high-yield recombinant expression system for glutamine synthetase of Mycobacterium tuberculosis together with a simple purification. The procedure allowed the structure of a complex with a phosphorylated form of the inhibitor methionine sulfoximine, magnesium, and ADP to be solved by molecular replacement and refined at 2.1-Å resolution. To our knowledge, this study provides the first reported structure for a taut form of the M. tuberculosis enzyme, similar to that observed for the Salmonella enzyme earlier. The phospho compound, generated in situ by an active enzyme, mimics the phosphorylated tetrahedral adduct at the transition state. Some differences in ligand interactions of the protein with both phosphorylated compound and nucleotide are observed compared with earlier structures; a third metal ion also is found. The importance of these differences in the catalytic mechanism is discussed; the results will help guide the search for specific inhibitors of potential therapeutic interest.

Maltose-binding proteins act as primary receptors in bacterial transport and chemotaxis systems. We report here crystal structures of the thermoacidostable maltose-binding protein from Alicyclobacillus acidocaldarius, and explore its modes of binding to maltose and maltotriose. Further, comparison with the structures of related proteins from Escherichia coli (a mesophile), and two hyperthermophiles (Pyrococcus furiosus and Thermococcus litoralis) allows an investigation of the basis of thermo- and acidostability in this family of proteins. The thermoacidophilic protein has fewer charged residues than the other three structures, which is compensated by an increase in the number of polar residues. Although the content of acidic and basic residues is approximately equal, more basic residues are exposed on its surface whereas most acidic residues are buried in the interior. As a consequence, this protein has a highly positive surface charge. Fewer salt bridges are buried than in the other MBP structures, but the number exposed on its surface does not appear to be unusual. These features appear to be correlated with the acidostability of the A. acidocaldarius protein rather than its thermostability. An analysis of cavities within the proteins shows that the extremophile proteins are more closely packed than the mesophilic one. Proline content is slightly higher in the hyperthermophiles and thermoacidophiles than in mesophiles, and this amino acid is more common at the second position of β-turns, properties that are also probably related to thermostability. Secondary structural content does not vary greatly in the different structures, and so is not a contributing factor.