Porcine kidney d-amino acid oxidase-derived R-amine oxidases with new substrate specificities.

An R-stereoselective amine oxidase and variants with markedly altered substrate specificity toward (R)-amines were generated from porcine d-amino acid oxidase (pkDAO), based on the X-ray crystallographic analysis of the wild-type enzyme. The new R-amine oxidase, a pkDAO variant (Y228L/R283G), acted on α-MBA and its derivatives, α-ethylbenzylamine, alkylamine, and cyclic secondary amines, totally losing the activities toward the original substrates, d-amino acids. The variant is enantiocomplementary to the flavin-type S-stereoselective amine oxidase variant from Aspergillus niger. Moreover, we solved the structure of pkDAO variants and successfully applied the obtained information to generate more variants through rational protein engineering, and used them in the synthesis of pharmaceutically attractive chiral compounds. The pkDAO variant Y228L/R283G and a variant I230A/R283G were used to synthesize (S)-amine and (R)-4-CBHA through deracemization, from racemic α-methylbenzylamine and benzhydrylamine, respectively, by selective oxidation of one of the enantiomers in the presence of a chemical reductant such as NaBH4. From a mechanistic point of view, we speculated that the imine intermediate, synthesized by oxidases or dehydrogenases, could be converted into primary α-aminonitrile by nucleophilic addition of cyanide in aqueous solutions. Nitriles and some unnatural amino acids were synthesized through a cascade reaction by oxidative cyanation reaction with the variant and a wide substrate specificity nitrilase.

Regulatory mechanism of the flavoprotein Tah18-dependent nitric oxide synthesis and cell death in yeast.

Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. The regulatory mechanism of NO generation in unicellular eukaryotic yeast cells is poorly understood due to the lack of mammalian and bacterial NO synthase (NOS) orthologues, even though yeast produces NO under oxidative stress conditions. Recently, we reported that the flavoprotein Tah18, which was previously shown to transfer electrons to the iron-sulfur cluster protein Dre2, is involved in NOS-like activity in the yeast Saccharomyces cerevisiae. On the other hand, Tah18 was reported to promote apoptotic cell death after exposure to hydrogen peroxide (H2O2). Here, we showed that NOS-like activity requiring Tah18 induced cell death upon treatment with H2O2. Our experimental results also indicate that Tah18-dependent NO production and cell death are suppressed by enhancement of the interaction between Tah18 and its molecular partner Dre2. Our findings indicate that the Tah18-Dre2 complex regulates cell death as a molecular switch via Tah18-dependent NOS-like activity in response to environmental changes.

Structural and computational analysis of peptide recognition mechanism of class-C type penicillin binding protein, alkaline D-peptidase from Bacillus cereus DF4-B.

Alkaline D-peptidase from Bacillus cereus DF4-B, called ADP, is a D-stereospecific endopeptidase reacting with oligopeptides containing D-phenylalanine (D-Phe) at N-terminal penultimate residue. ADP has attracted increasing attention because it is useful as a catalyst for synthesis of D-Phe oligopeptides or, with the help of substrate mimetics, L-amino acid peptides and proteins. Structure and functional analysis of ADP is expected to elucidate molecular mechanism of ADP. In this study, the crystal structure of ADP (apo) form was determined at 2.1 Å resolution. The fold of ADP is similar to that of the class C penicillin-binding proteins of type-AmpH. Docking simulations and fragment molecular orbital analyses of two peptides, (D-Phe)4 and (D-Phe)2-(L-Phe)2, with the putative substrate binding sites of ADP indicated that the P1 residue of the peptide interacts with hydrophobic residues at the S1 site of ADP. Furthermore, molecular dynamics simulation of ADP for 50 nsec suggested that the ADP forms large cavity at the active site. Formation of the cavity suggested that the ADP has open state in the solution. For the ADP, having the open state is convenient to bind the peptides having bulky side chain, such as (D-Phe)4. Taken together, we predicted peptide recognition mechanism of ADP.

Mutagenesis of an Asn156 Residue in a Surface Region of S-Selective Hydroxynitrile Lyase from Baliospermum montanum Enhances Catalytic Efficiency and Enantioselectivity.

The S-selective hydroxynitrile lyase from Baliospermum montanum (BmHNL) has broad substrate specificity toward aromatic substrates as well as high temperature stability, although with low enantioselectivity and specific activity. To expand the industrial application of this enzyme, we improved its enantioselectivity and specific activity toward (S)-mandelonitrile by mutagenesis. The specific activity of the BmHNL H103C/N156G mutant for (S)-mandelonitrile production was raised to 154 U mg-1 (WT BmHNL: 52 U mg-1 ). The enantiomeric excess was increased to 93 % (WT BmHNL: 55 %). The kinetic analysis revealed Km for (R)-mandelonitrile and kcat for (S)-mandelonitrile increased by the mutation at Asn156, thus contributing to the increase in enantiomeric excess. This is the first report on an improvement in catalytic efficiency and enantiomeric excess of BmHNL for (S)-mandelonitrile synthesis by random and site-directed mutagenesis.

The flavoprotein Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells.

Nitric oxide (NO) is a ubiquitous signaling molecule involved in the regulation of a large number of cellular functions. In the unicellular eukaryote yeast, NO may be involved in stress response pathways, but its role is poorly understood due to the lack of mammalian NO synthase (NOS) orthologues. Previously, we have proposed the oxidative stress-induced l-arginine synthesis and its physiological role under stress conditions in yeast Saccharomyces cerevisiae. Here, our experimental results indicated that increased conversion of l-proline into l-arginine led to NO production in response to elevated temperature. We also showed that the flavoprotein Tah18, which was previously reported to transfer electrons to the Fe-S cluster protein Dre2, was involved in NO synthesis in yeast. Gene knockdown analysis demonstrated that Tah18-dependent NO synthesis confers high-temperature stress tolerance on yeast cells. As it appears that such a unique cell protection mechanism is specific to yeasts and fungi, it represents a promising target for antifungal activity.