Crystal Structure ofd-Hydantoinase fromBurkholderia pickettiiat a Resolution of 2.7 Angstroms: Insights into the Molecular Basis of Enzyme Thermostability

Abstract

D-Hydantoinase (D-HYD) is an industrial enzyme that is widely used in the production of D-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides. This report describes the crystal structure of D-hydantoinase from Burkholderia pickettii (HYD Bp ) at a 2.7-Å resolution. The structure of HYD Bp consists of a core (␣/␤) 8 triose phosphate isomerase barrel fold and a ␤-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues. Although HYD Bp shares only moderate sequence similarity with D-HYDs from Thermus sp. (HYD Tsp ) and Bacillus stearothermophilus (HYD Bst ), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar. Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity. Structural comparison has revealed insights into the molecular basis of the differential thermostability of D-HYDs. The more thermostable HYD Tsp contains more aromatic residues in the interior of the structure than HYD Bp and HYD Bst . Changes of large aromatic residues in HYD Tsp to smaller residues in HYD Bp or HYD Bst decrease the hydrophobicity and create cavities inside the structure. HYD Tsp has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYD Bp and HYD Bst . Besides, HYD Tsp also contains more rigid Pro residues. These factors are likely to make major contributions to the varying thermostability of these enzymes. This information could be exploited in helping to engineer more thermostable mesophilic enzymes.Dihydropyrimidinases (EC 3.5.2.2) are a family of enzymes that catalyze the reversible hydrolytic ring opening of the amide bond in five-or six-membered cyclic diamides (54, 62). In humans, they are responsible for the hydrolysis of dihydropyrimidines at the second step of reductive catabolism of pyrimidines. The homologous enzymes in microorganisms are hydantoinases (HYDs) which catalyze the hydrolysis of 5-substituted hydantoins to the enantiomerically pure Ncarbamyl amino acids. The latter can be converted chemically or enzymatically to the corresponding optically pure amino acids (50, 54, 64). In the biotechnology industry, HYD is widely used in the production of D-amino acids, including D-p-hydroxyphenylglycine, which are the precursors for semisynthesis of antibiotics, peptide hormones, pyrethroids, and pesticides (3,49,53,54). Depending on their stereoselectivities and specificities on substrates, HYDs are often classified as D-, L-, or non-enantio specific (47, 67).One major consideration in the application of enzymes for biocatalysis is thermostability. Screening of various thermophiles has successfully discovered several thermostable D-HYDs from Thermus sp. CBS30380 and Lu1220 (28), and Bacillus stearothermophilus SD-1 (38) and GH-2 (51). Substantial efforts...

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: A biophysical characterization

“…Dihydropyrimidinases have been found in diverse organisms, such as bacteria, animal, yeast and plants [4][5][6][7][8]. Several of these enzymes, with different stereoselectivities and substrate specificities, have been used in industrial bioconversion of optically pure D-amino acids with 5-monosubstituted hydantoins as substrates [9,10].…”

Divergent Functions Through Alternative Splicing: The Drosophila CRMP Gene in Pyrimidine Metabolism, Brain, and Behavior

“…All members of the DHP/CRMP family function in vivo as homotetramers and, possibly, heterotetramers (Wang and Strittmatter 1997;Deo et al 2004); their resolved structures are extraordinarily similar (Abendroth et al 2002a,b;Xu et al 2003;Deo et al 2004;Lohkamp et al 2006;Stenmark et al 2007). A key functional distinction among these proteins is that DHPs are zinc-coupled dihydropyrimidine hydrolases, whereas no comparable hydrolase activity has been shown for vertebrate CRMPs, which lack one or more essential zinc-binding site residues found in DHP proteins (Hamajima et al 1998;Wang and Strittmatter 1997;Takemoto et al 2000).…”

“…DHP is found in most eukaryotes and catalyzes the second step of reductive pyrimidine catabolism (reviewed in Schnackerz and Dobritzch 2008). On the other hand, CRMPs appear to be limited to nervous systems of metazoans, where they mediate a variety of processes: semaphorin signal transduction in axonal growth cones, cytoskeletal dynamics, neuronal polarity, and modulation of neurotransmitter release (reviewed in Schmidt and Strittmatter 2007;Hou et al 2008;Chi et al 2009;Yamashita and Goshima 2012).All members of the DHP/CRMP family function in vivo as homotetramers and, possibly, heterotetramers (Wang and Strittmatter 1997;Deo et al 2004); their resolved structures are extraordinarily similar (Abendroth et al 2002a,b;Xu et al 2003;Deo et al 2004;Lohkamp et al 2006;Stenmark et al 2007). A key functional distinction among these proteins is that DHPs are zinc-coupled dihydropyrimidine hydrolases, whereas no comparable hydrolase activity has been shown for vertebrate CRMPs, which lack one or more essential zinc-binding site residues found in DHP proteins (Hamajima et al 1998;Wang and Strittmatter 1997;Takemoto et al 2000).…”

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

“…Second, the putative active site water molecule, which is likely to be present at the product acetate ACT1 O 1 position, lies significantly closer to the zinc ion at the ␣ 1 subsite (1.95 Å) than that at the ␤ site (2.84 Å). This is one striking difference between the active site here and those in other binuclear zinc hydrolases such as the ␣␤ members, where the water/hydroxide ion is almost symmetrically located between the two metal ions (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Therefore, the inhibitory metal ion at the ␣ site may hold the active site water tightly and thus perturb the stereochemical arrangements required for enzyme catalysis as it does in bovine carboxypeptidase A (38).…”

“…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)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Murine adenosine deaminase and Escherichia coli cytosine deaminase belong to the ␣ subset (16,17).…”