Evolution of enzymatic activities in the enolase superfamily: N-succinylamino acid racemase and a new pathway for the irreversible conversion of D- to L-amino acids.
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.
Center for Biophysics and Computational Biology and Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Ayano
A
Sakai
Dao Feng
DF
Xiang
Chengfu
C
Xu
Ling
L
Song
Wen Shan
WS
Yew
Frank M
FM
Raushel
John A
JA
Gerlt
D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000378:metabolism; D000591:Amino Acid Isomerases / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D005075:Biological Evolution; D007700:Kinetics; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D013386:Succinates / Q000378:metabolism
D000214:Acyl Coenzyme A; D000596:Amino Acids; D013386:Succinates; D000217:Acyltransferases; D010751:Phosphopyruvate Hydratase; D000591:Amino Acid Isomerases; C510484:N-succinylamino acid racemase, Geobacillus kaustophilus