The Functional Role of the Binuclear Metal Center in d-Aminoacylase

Abstract

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: ␣␤-binuclear, ␣-mononuclear, ␤-mononuclear, and metal-independent subsets. The D-aminoacylase from Alcaligenes faecalis DA1 belongs to the ␤-mononuclear subset due to the fact that the catalytically essential , revealing that elimination of the ␤ site changes the coordination geometry of the ␣ ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the Daminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the ␤ site, but inhibited by the subsequent binding of the second ion at the ␣ site.D-Aminoacylase (EC 3.5.1.81) is an attractive candidate for production of D-amino acids through its catalysis of the deacetylation of N-acetyl-D-amino acids. Since D-amino acids are intermediates in the preparation of antibiotics, pesticides, and bioactive peptides, D-aminoacylase has commercial importance for the optical resolution of N-acetyl-DL-amino acids (1-2). The crystal structure of the 483-residue D-aminoacylase from Alcaligenes faecalis DA1 revealed that the enzyme is comprised of a small ␤ barrel and a catalytic TIM barrel with a unique 63-residue insertion (3). The structural similarity and the conserved metal ligands of four histidines and one aspartate suggest that D-aminoacylase belongs to the TIM barrel metal-dependent hydrolase superfamily. This superfamily includes a variety of enzymes with highly diverse substrates and has been coined an "evolutionary treasure" (4).To date, crystal structures of 14 members in the superfamily have been determined. As more structures are solved, more divergences in the metal centers and in the ␤-strand packing of the TIM barrel are observed (Fig. 1). Based on the binding site(s) of the catalytically essential metal ion(s), we classify these members into four types: ␣␤-binuclear (␣␤), ␣-mononuclear (␣), ␤-mononuclear (␤), and metal-independent subsets. Phosphotriesterase (homology protein), urease, dihydroorotase, renal dipeptidase, isoaspartyl didpeptidase, and dihydropyrimidinase comprise the ␣␤ subset (5-15). Murine adenosine deaminase and Escherichia coli cytosine deaminase belong to the ␣ subset (16, 17). D-Aminoacylase belongs to the ␤ subset due to the essential Zn 2ϩ binding tightly at the ␤ site, even though it possesses a weak ␣ binding site (3). In contrast, the activity assay and the crystal structure of uronate isomerase show that this enzyme does not display any specific metal requirement and thus belongs to the metal-independent subset (18,19).The functional requirement for the different metal centers is still unclear. Interestingly, Bacillus subtilis N-acetylglucosamine-6-phosphate deacetylase ...

Promiscuous Acylases‐Catalyzed Markovnikov Addition of N‐Heterocycles to Vinyl Esters in Organic Media

“…Ligation of the inhibitory zinc ion by the highly conserved residues Asp 366 , His 67 and His 69 in the active site would lower their pK a values and/or hold the nucleophile to perturb the proton shuttle and intermediate stabilization. [12] The inScheme 1. Markovnikov addition of imidazoles to vinyl esters.…”

Crystal Structures of Nonoxidative Zinc-dependent 2,6-Dihydroxybenzoate (γ-Resorcylate) Decarboxylase from Rhizobium sp. Strain MTP-10005

“…Because these enzymes are members of the recently identified TIM-barrel metallo-dependent hydrolase superfamily, GRDC is classified as a member of this superfamily. The superfamily is mainly divided into two subgroups depending on a bi-metal or mono-metal enzyme (47,48), and GRDC thus belongs to the mono-metal-dependent hydrolase subgroup together with Fe 3ϩ -dependent cytosine deaminase (42) and Zn 2ϩ -dependent adenosine deaminase (46). Possibly, the active site structure of GRDC is more similar to that of adenosine deaminase than cytosine deaminase in terms of the mono-Zn 2ϩ binding.…”

Crystal Structure of Bacillus subtilis Guanine Deaminase

“…For example, the Bacillus subtilis GD (bGD) has been shown to be inducible with purines as nitrogen sources (3,4). On the other hand, mammalian, insect, fungal, and some bacterial GDs belong to the TIM barrel metallohydrolase superfamily (5,6). GD is the only identified enzyme in mammals that can directly deaminate a base, and its diagnostic usefulness has been well documented, for example, in the detection of hepatoma and the identification of donor blood infected with the hepatitis C virus (7,8).…”