Crystal Structures of Yeast β-Alanine Synthase Complexes Reveal the Mode of Substrate Binding and Large Scale Domain Closure Movements

Abstract

␤-Alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu 159 and Arg 322 as crucial for catalysis and His 262 and His 397 as functionally important but not essential. We determined the crystal structures of wild-type ␤-alanine synthase in complex with the reaction product ␤-alanine, and of the mutant E159A with the substrate N-carbamyl-␤-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a ϳ30°rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.

Potential Application ofN-Carbamoyl-β-Alanine Amidohydrolase fromAgrobacterium tumefaciensC58 for β-Amino Acid Production

“…None of the mutants showed activity using the standard assay, although only the lysine mutant enzyme secondary structure elements were unaltered when analyzed by far-UV circular dichroism (data not shown). The R291K mutation suggests how essential this residue is in substrate binding, supporting previous results found for other enzymes (19,24).…”

“…The ␤car At model was obtained by the Swiss-Model server (34), using the substrate-bound structure of Saccharomyces kluyveri NC␤AA (Sk␤as) (PDB accession no. 2V8H_A), which has been solved at 2.0 Å, as a template (19). The stereochemical geometry of the final model was validated by PROCHECK (16) and WHATCHECK (13), included in the model assessment tools of the Swiss-Model server.…”

“…The involvement and roles of several residues comprising the catalytic center of L-N-carbamoylases and Sk␤as have been proved (19,24), and they are completely conserved in ␤car At (Fig. 1), in which Arg291 would be the key residue for recognition of the substrate carboxyl group.…”

Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

“…Unexpectedly, a highly conserved arginine in L-carbamoylases has been proved critical for the dimerization of the enzyme, and thus for enzymatic activity. Comparative studies revealed striking characteristics of the different "dimerization" domains of the peptidase M20/M25/ M40 family, in agreement with previous evolutionary hypotheses on this family of enzymes (3,37) and suggesting new evolutionary considerations. Finally, an alternative reaction mechanism dependent on only one metal atom can be inferred from the experimental data.…”

“…In fact, the Arg and His residues are strictly conserved. Based on the literature available for homologous enzymes, the conserved Arg residue is or might be necessary for substrate binding in ␤-alanine synthase (4,37,38), EcAam (2), peptidase V (PepV) (31), carnosinase (55), IAA-amino acid hydrolase homolog 2 (ILL2) (7), peptidase D (PepD) (9), DapE (46), hAcy1 (35), peptidase T (PepT) (6,24), and metallopeptidase (Sapep) (20). Several homologous structures present different molecules bound to these residues, namely, the structures under PDB IDs 1VIX, 1FNO, 3IC1, 3IFE, and 2QYV.…”