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

• Published in: Biochemistry (Easton) Vol. 45; no. 14; pp. 4455 - 4462
• Main Authors: Sakai, Ayano, Xiang, Dao Feng, Xu, Chengfu, Song, Ling, Yew, Wen Shan, Raushel, Frank M, Gerlt, John A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 11-04-2006
• Subjects: Acyl Coenzyme A - metabolism; Acyltransferases - metabolism; Amino Acid Isomerases - metabolism; Amino Acids - metabolism; Biological Evolution; Kinetics; Phosphopyruvate Hydratase - metabolism; Succinates - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi060230b

Members of the mechanistically diverse enolase superfamily catalyze reactions that are initiated by abstraction of the α-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 (β/α)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 β-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.