Strukturelle Charakterisierung zweier Schlüsselenzyme aus fermentativen Abbauwegen anaerober Bakterien

Enamidase The hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to 2-formylglutarate is a central step in the catabolism of nicotinate in several Clostridia and Proteobacteria. This reaction is cata-lyzed by the novel enzyme enamidase (EC 3.5.2.18), a new member of the amidohydrolase superfamily as indicated by its unique reaction, sequence comparison, and the stoichiometric binding of metal ions. A hallmark of enamidase is its capability to catalyze a two-step reac-tion: the initial decyclization of 1,4,5,6-tetrahydro-6-oxonicotinate leading to 2-(enamine)glutarate followed by an additional hydrolysis step yielding (S)-2-formylglutarate. This study presents the crystal structure of enamidase from Eubacterium barkeri at 1.9 Å, pro-viding a structural basis for catalysis and suggesting a mechanism for its exceptional activity and enantioselectivity. The enzyme forms a 222-symmetric tetramer built up by a dimer of dimers. Each enamidase monomer consists of a composite beta-sandwich domain compris-ing the N- and C-termini of the polypeptide chain and an (α/β)8-TIM-barrel domain harbor-ing the active site. With its catalytic binuclear metal center comprising both zinc and iron ions, enamidase represents a special case of subtype II amidohydrolases. Decarboxylase subunit of the sodium ion pump glutaconyl-CoA decarboxylase Glutaconyl-CoA decarboxylase (Gcd, EC 4.1.1.70) couples the biotin-dependent decarboxyla-tion of glutaconyl-CoA with the generation of an electrochemical Na+ gradient. As an inte-gral membrane enzyme Gcd comprises four different subunits: the α-subunit (GcdA) cata-lyzing the transfer of the carboxylate of glutaconyl-CoA to the biotinylated γ subunit (GcdC), the membrane resident β-subunit (GcdB) responsible for the subsequent decarboxy-lation of carboxybiotin coupled to Na+ translocation and a small δ-subunit (GcdD) of yet un-known function. Co-crystallization of the decarboxylase subunit GcdA with the substrate glutaconyl-CoA, the product crotonyl-CoA and the substrate analogue glutaryl-CoA, respec-tively, resulted in a high resolution model for quaternary structure and catalysis of the en-zyme revealing remarkable structural changes upon substrate binding. On the basis of these data a new asymmetric model for the intact Gcd complex is presented in which GcdA is as-sociated as a tetramer crisscrossed by a network of solvent filled channel