The transcarboxylase multienzyme complex: Structural studies of the components of a 1.2 million-Dalton carboxylation machine

Transcarboxylase from Propionibacterium shermanii is a 1.2 million dalton multienzyme complex that couples two carboxylation reactions, transferring CO2- from methylmalonyl-CoA to pyruvate, yielding propionyl-CoA and oxaloacetate. We have solved the crystal structures of the transcarboxylase central 12S hexameric core and the outer 5S dimeric subunit. The 1.9 A resolution crystal structure of the 12S subunit, which catalyzes the first carboxylation reaction, has been solved bound to its substrate methylmalonyl-CoA. Overall, the structure reveals two stacked trimers related by 2-fold symmetry, and a domain duplication in the monomer. In the active site, the labile carboxylate group of methylmalonyl-CoA is stabilized by interaction with two peptide NH groups located at the N-termini of alpha-helices. 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 beta-subunit has 50% sequence identity with 12S. A homology model of the propionyl-CoA carboxylase beta-subunit, based on this 12S crystal structure, provides new insight into the propionyl-CoA carboxylase mechanism, its oligomeric structure, and the molecular basis of mutations responsible for enzyme deficiency in propionic acidemia.; Crystal structures of the 5S metalloenzyme subunit, which catalyzes the second carboxylation reaction, have been solved in apo form and bound to its substrate pyruvate, product oxaloacetate, or inhibitor 2-ketobutyrate. The structure reveals a dimer of beta 8alpha8 barrels with an active site cobalt ion coordinated by a carbamylated lysine, except in the oxaloacetate complex when the product's carboxylate group serves as a ligand instead. 5S and human pyruvate carboxylase (PC), an enzyme crucial to gluconeogenesis, catalyze similar reactions. A 5S crystal structure based homology model of the PC carboxyltransferase domain indicates a conserved mechanism and explains the molecular basis of mutations in lactic acidemia. PC disease mutations reproduced in 5S result in a similar decrease in carboxyltransferase activity and crystal structures with altered active sites. Finally, we have computationally docked our 5S and 12S crystal structures, and the partial NMR structure of 13S, to generate a TC holo multienzyme complex model consistent with electron microscopic studies, giving the most detailed look to date at this 1.2 million Dalton carboxylation machine.