Crystal structure of the hydrogenase maturating endopeptidase HYBD from Escherichia coli

The maturation of [NiFe] hydrogenases includes formation of the nickel meta llocenter, proteolytic processing of the metal center carrying large subuni t, and its assembling with other hydrogenase subunits. The hydrogenase matu rating enzyme HYBD from Escherichia coli, a protease of molecular mass 17.5 kDa, specifically cleaves off a 15 amino acid peptide from the C terminus of the precursor of the large subunit of hydrogenase 2 in a nickel-dependen t manner. Here we report the crystal structure of HYBD at 2.2 Angstrom reso lution. It consists of a twisted five-stranded beta-sheet surrounded by fou r and three helices, respectively, on each side. A cadmium ion from the cry stallization buffer binds to the proposed nickel-binding site and is penta- coordinated by Glu16, Asp62, His93, and a water molecule in a pseudo-tetrag onal arrangement. HYBD is topologically related to members of the metzincin s superfamily of zinc endoproteinases, sharing the central beta-sheet and t hree helices. In contrast to the metzincins, the metal-binding site of HYBD is localized at the C-terminal end of the beta-sheet. Three helical insert ions unique to HYBD pack against one side of the sheet, build up the active site cleft, and provide His93 as ligand to the metal. From this structure, we derive molecular clues into how the protease HYBD is involved in the hy drogenase maturation process. (C) 1999 Academic Press.