Members of the mechanistically diverse
enolase superfamily catalyze reactions that are initiated by abstraction of the alpha-
proton of a carboxylate
anion to generate an enolate
anion intermediate that is stabilized by coordination to a Mg2+ ion. The catalytic groups,
ligands for an essential Mg2+ and
acid/base catalysts, are located in the (beta/alpha)8-barrel domain of the bidomain
proteins. The assigned physiological functions in the muconate lactonizing
enzyme (MLE) subgroup (Lys
acid/base catalysts at the ends of the second and sixth beta-strands in the barrel domain) are cycloisomerization (MLE),
dehydration (
o-succinylbenzoate synthase; OSBS), and epimerization (L-Ala-D/L-Glu
epimerase). We previously studied a putatively promiscuous member of the MLE subgroup with uncertain physiological function from Amycolatopsis that was discovered based on its ability to catalyze the racemization of N-acylamino
acids (N-acylamino
acid racemase; NAAAR) but also catalyzes the OSBS reaction [OSBS/NAAAR; Palmer, D. R., Garrett, J. B., Sharma, V., Meganathan, R., Babbitt, P. C., and Gerlt, J. A. (1999) Biochemistry 38, 4252-4258]. In this manuscript, we report functional characterization of a homologue of this
protein encoded by the genome of Geobacillus kaustophilus as well as two other
proteins that are encoded by the same operon, a divergent member of the Gcn5-related N-
acetyltransferase (GNAT) superfamily of
enzymes whose members catalyze the transfer an acyl group from an
acyl-CoA donor to an
amine acceptor, and a member of the M20
peptidase/
carboxypeptidase G2 family. We determined that the member of the GNAT superfamily is
succinyl-CoA:D-
amino acid N-succinyltransferase, the member of the
enolase superfamily is N-succinylamino
acid racemase (NSAR), and the member of the M20
peptidase/
carboxypeptidase G2 family is N-succinyl-L-
amino acid hydrolase. We conclude that (1) these
enzymes constitute a novel, irreversible pathway for the conversion of D- to L-
amino acids and (2) the NSAR reaction is a new physiological function in the MLE subgroup. The NSAR is also functionally promiscuous and catalyzes an efficient OSBS reaction; intriguingly, the operon for
menaquinone biosynthesis in G. kaustophilus does not encode an OSBS, raising the possibility that the NSAR is a bifunctional
enzyme rather than an accidentally promiscuous
enzyme.