Abstract
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.
