Formyl-coenzyme : a transferase, structure and enzymatic mechanism

Sammanfattning: Background: Formyl-CoA transferase (Frc) is the first enzyme in a two enzyme pathway responsible for oxalate degradation in Oxalobacter formigenes. This bacterium is a constitutive part of human intestinal flora. Its role as an oxalate scavenger is very important, reports have shown a strong connection between the disappearance of 0. formigenes and the appearance of disorders related to oxalate accumulation (e.g. kidney stones, renal failure, cardiac disorders). Frc is a protein of 428 amino acids and belongs to a newly identified third family of CoA transferases where no structural characterisation was previously available. Moreover an enzymatic mechanism has not been proposed for any member of family III of CoA transferases. Results: Frc has been purified and crystallised; subsequently the three dimensional structure of the enzyme was elucidated by X-ray crystallography to 2.2 A resolution. The monomer structure consists of an N-terminal Rossmann fold-like domain, followed by a small domain connected with the N-terminal domain by a long helix. The C-terminal part of Frc is an elongated 70 amino acids loop that interacts with the Rossmann fold-like domain; thus the monomer is shaped as a ring. The homodimer displays a protein fold never observed before, the two subunits are interlocked as two rings of a chain. The structure of Frc in complex with coenzyme A was solved in order to pinpoint the active site. CoA binds to the Rossmann fold-like domain at the nucleotide binding Pap motif; nonetheless CoA binds to it in a very different way. Frc has been characterised kinetically and three mutants of the putative catalytic amino acid Asp169 have been analysed structurally and kinetically. These mutants are almost or totally inactive confirming the importance of Asp169. The structure of Frc in complex with its product oxalyl-CoA has been elucidated. It shows the oxalyl moiety covalently bound to Asp169 as oxalyl-aspartic anhydride. This confirms the existence of anhydrides as intermediates of the reaction and that Asp 169 is the amino acid performing the nucleophilic attack on formyl-CoA.