Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis

A large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 å. The Rv0130 monomer features a single hotdog fold composed of a highly curved β‐sheet on top of a long and a short α‐helix. Two monomers in turn pack to form a double‐hotdog‐folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R ‐specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans ‐2‐enoyl‐coenzyme A moiety with a k cat of 1.1 × 10 2 sec −1 . The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site‐directed mutagenesis. In contrast to many hotdog‐folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate‐binding channels of long‐chain eukaryotic hydratase 2 enzymes.