Yeast β-Alanine Synthase Shares a Structural Scaffold and Origin with Dizinc-dependent Exopeptidases

Abstract

␤-Alanine synthase (␤AS) is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of pyrimidine bases, including several anticancer drugs. In eukaryotes, ␤ASs belong to two subfamilies, which exhibit a low degree of sequence similarity. We determined the structure of ␤AS from Saccharomyces kluyveri to a resolution of 2.7 Å. The subunit of the homodimeric enzyme consists of two domains: a larger catalytic domain with a dizinc metal center, which represents the active site of ␤AS, and a smaller domain mediating the majority of the intersubunit contacts. Both domains exhibit a mixed ␣/␤-topology. Surprisingly, the observed high structural homology to a family of dizinc-dependent exopeptidases suggests that these two enzyme groups have a common origin. Alterations in the ligand composition of the metal-binding site can be explained as adjustments to the catalysis of a different reaction, the hydrolysis of an N-carbamyl bond by ␤AS compared with the hydrolysis of a peptide bond by exopeptidases. In contrast, there is no resemblance to the three-dimensional structure of the functionally closely related N-carbamyl-D-amino acid amidohydrolases. Based on comparative structural analysis and observed deviations in the backbone conformations of the eight copies of the subunit in the asymmetric unit, we suggest that conformational changes occur during each catalytic cycle.In most living organisms, the degradation of uracil and thymine is achieved by the three-step reaction sequence of the reductive pyrimidine catabolic pathway. The first and rate-limiting enzyme is dihydropyrimidine dehydrogenase (dihydrouracil dehydrogenase (NADP ϩ ), EC 1.3.1.2), which catalyzes the NADPHdependent reduction of uracil and thymine to the corresponding dihydropyrimidines (Scheme 1). In the second step, dihydropyrimidinase (EC 3.5.2.2) generates N-carbamyl-␤-alanine and N-carbamyl-␤-aminoisobutyric acid, respectively, via reversible

Cell Division Defects of Schizosaccharomyces pombe liz1 − Mutants Are Caused by Defects in Pantothenate Uptake

“…4). It is not clear if ureidopropionate is converted to ␤-alanine by spontaneous hydrolysis (8) or if the identification of the S. pombe ␤-alanine synthase is prevented by the sequence diversity known to occur in eukaryotic ␤-alanine synthases (16,24).…”

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

“…Among the five residues involved in the binuclear metal center in the structures known to date (Table 2), the M2 binding residues (BsLcar Glu 125 and His 380 ) are totally conserved, whereas M1 binding residues present a higher level of divergence (His 79 is also fully conserved, but His 189 may appear as a Glu or Asp residue). This divergence was explained for the homologous EcAam and Sk␤as enzymes, as a His residue would allow a lower pK a of the Zn-bound water than that with a negatively charged lateral chain (Glu or Asp) (2,38). In fact, the His residue would allow an increased nucleophilicity of the attacking hydroxyl, which was hypothesized as necessary to hydrolyze a carbamoyl moiety compared to a peptide bond due to the better resonance stabilization of the ureido group (2,38).…”

“…This divergence was explained for the homologous EcAam and Sk␤as enzymes, as a His residue would allow a lower pK a of the Zn-bound water than that with a negatively charged lateral chain (Glu or Asp) (2,38). In fact, the His residue would allow an increased nucleophilicity of the attacking hydroxyl, which was hypothesized as necessary to hydrolyze a carbamoyl moiety compared to a peptide bond due to the better resonance stabilization of the ureido group (2,38). Although the same hypothesis would support a BsLcar mechanism dependent on a single cation (as M1 is coordinated through two His residues, like the case for EcAam and Sk␤as) ( Table 2; see Fig.…”

“…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.…”

Crystallization and preliminary X-ray data analysis of β-alanine synthase fromDrosophila melanogaster

“…Phylogenetic analyses revealed that they can be assigned to two subfamilies (Gojković et al, 2001), with the majority of the eukaryotic enzymes, including those from human, calf, rat and fruit fly, forming one of them. The enzyme from the yeast Saccharomyces kluyveri, to date the only AS with known crystal structure Lundgren et al, 2003), shows no sequence similarity to this group but is related to bacterial N-carbamyl-l-amino-acid amidohydrolases, hence forming a separate subfamily. It is a dimer of identical subunits comprising two domains with / topology, the larger of which contains a di-zinc centre that is essential for catalytic activity.…”