The structure of the C–C bond hydrolase MhpC provides insights into its catalytic mechanism

摘要: 2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) is a 62 kDa homodimeric enzyme of the phenylpropionate degradation pathway Escherichia coli. The 2.1 A resolution X-ray structure native determined from orthorhombic crystals confirms that it member ?/? hydrolase fold family, comprising eight ?-strands interconnected by loops and helices. 2.8 co-crystallised with non-hydrolysable substrate analogue 2,6-diketo-nona-1,9-dioic (DKNDA) location active site in buried channel including Ser110, His263 Asp235, postulated contributors to serine protease-like catalytic triad homologous enzymes. It appears ligand binds two separate orientations. In first, C6 keto group inhibitor forms hemi-ketal adduct Ser110 side-chain, C9 carboxylate interacts, via intermediacy water molecule, Arg188 at one end site, while C1 comes close His114 other end. second orientation, These arrangements implicated or as plausible catalysis initial enol/keto tautomerisation but lack conservation amongst related enzymes mutagenesis results suggest residue involved. Variability quality electron density for molecules crystal asymmetric unit correlate alternative positions side-chain His114. This might arise half-site occupation dimeric reflect apparent dissociation approximately 50% intermediate during cycle.