W0131

Transcarboxylase Multienzyme Complex: MMCoA Bound 12S Hexamer at 2.0 Å Resolution and Towards 5S Subunit MAD Phasing. Pamela H. Roberts^1,3, Yan-fei Wang¹, Rosa E. Rivera-Hainaj⁴, Xiaojing Zheng⁴, Run Zheng⁴, Marianne Pustai-Carey⁴, Focco van den Akker², Paul R. Carey⁴, Vivien C. Yee^1,3, Depts. of ¹Molecular Cardiology and of ²Molecular Biology and Center for Structural Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH; Depts. of ³Pharmacology and of ⁴Biochemistry, Case Western Reserve Univ., Cleveland, OH.

Human biotin-dependent carboxylases play central roles in a variety of metabolic processes. Deficiencies of two of these enzymes, pyruvate carboxylase and propionyl-CoA carboxylase, result in the diseases lactic acidemia and propionic acidemia, respectively. Transcarboxylase (TC) from Propionibacterium shermanii has long functioned as a powerful model system for the study of biotin-dependent carboxylases. TC is a 1.2 million dalton multi-enzyme complex containing 30 polypeptide chains: a catalytic 336 kDa 12S hexameric core; six catalytic 116 kDa 5S dimers; and twelve 12 kDa 1.3S biotinylated linkers. TC transfers CO₂ from methylmalonyl-coenzyme A (MMCoA) to pyruvate, producing propionyl-CoA (PCoA) and oxaloacetate. From electron microscopy, the 5S dimers form two outer trimeric rings at opposing ends of a central 12S hexamer. We have recently crystallized selenomethionine derivatized 5S for MAD phasing. In addition, the 2.0 Å resolution crystal structure of the central 12S hexameric core has been solved bound to MMCoA. The structure reveals two stacked trimers related by 2-fold symmetry, and domain duplication in the monomer. The 12S domains are structurally similar to the crotonase /isomerase superfamily and the 12S reaction is similar to that of human propionyl-CoA carboxylase, whose β_subunit has 50% sequence identity with 12S. A homology model of the propionyl-CoA carboxylase β_subunit, based on this 12S crystal structure, provides insight into the molecular basis of mutations responsible for enzyme deficiency in propionic acidemia.