ABSTRACT
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.
Subject(s)
D-Amino-Acid Oxidase/chemistry , Kidney/enzymology , Protein Engineering , Amino Acids , Animals , Substrate Specificity , SwineABSTRACT
Fluidic chip fabrication technologies using three-dimensional (3D) printing have received broad attention recently. Herein, we describe a new method for fabricating polydimethylsiloxane (PDMS) fluidic chips using a 3D-printed polyvinyl alcohol (PVA) or acrylonitrile butadiene styrene (ABS) template and polymer coating. In this method, polyethylene glycol (PEG) was coated on the 3D-printed template. This coated template was immersed in liquid PDMS, and subsequently the PDMS was cured. Space can be created between the template and PDMS by removing this liquid PEG from the channel. This space renders template removal easier. A flow path is formed by dissolving the template with a solvent. These PDMS chips are used for flow injection measurement.
ABSTRACT
Amino acid oxidases are an important class of enzymes that mostly participate in the oxidation of amino acids using FAD as a cofactor. Many of them function in the catabolism of amino acids with wider substrate specificities. On the other hand, based on the recent, successful use of the enzymes for diagnoses with new cofactor and mechanism, highly selective enzymes have been screened from Nature, and many new enzymes have been discovered and further characterized by X-ray crystallography. As a result of the screening for amino acid oxidases with biosynthetic or antibiotic functions, l-Trp oxidase, l-Lys oxidases, and Gly oxidase have been found. The pyridoxal phosphate-dependent l-Arg oxidase has the intriguing new activity of hydroxylating unactivated CC bonds. A new amine oxidase was created by the protein engineering of d-amino acid oxidase. Recent developments in the characterization of amino acid oxidases and their applications are summarized.
Subject(s)
D-Amino-Acid Oxidase/metabolism , Amino Acids/metabolism , Crystallography, X-Ray , D-Amino-Acid Oxidase/chemistry , Oxidation-Reduction , Protein Engineering , Substrate SpecificityABSTRACT
Discovery and development of enzymes for the synthesis of chiral amines have been a hot topic for basic and applied aspects of biocatalysts. Based on our X-ray crystallographic analyses of porcine kidney D-amino acid oxidase (pkDAO) and its variants, we rationally designed a new variant that catalyzed the oxidation of (S)-4-Cl-benzhydrylamine (CBHA) from pkDAO and obtained it by functional high-throughput screening with colorimetric assay. The variant I230A/R283G was constructed from the variant R283G which had completely lost the activity for D-amino acids, further gaining new activity toward (S)-chiral amines with the bulky substituents. The variant enzyme (I230A/R283G) was characterized to have a catalytic efficiency of 1.85â s-1 for (S)-CBHA, while that for (R)-1-phenylethylamine was diminished 10-fold as compared with the Y228L/R283G variant. The variant was efficiently used for the synthesis of (R)-CBHA in 96 % ee from racemic CBHA by the deracemization reaction in the presence of reducing agent such as NaBH4 in water. Furthermore, X-ray crystallographic analysis of the new variant complexed with (S)-CBHA, together with modelling study clearly showed the basis of understanding the structure-activity relationship of pkDAO.
ABSTRACT
Elucidation of the molecular mechanism of amine oxidases (AOx) will help to extend their reactivity by rational design and their application to deracemization of various amine compounds. To date, several studies have been performed on S-selective AOx, but relatively few have focused on R-selective AOx. In this study, we sought to elucidate the mechanism of pkAOx, an R-selective AOx that we designed by introducing the Y228L and R283G mutations into d-amino acid oxidase from pig kidney. Four crystal structures of the substrate-bound protein and first-principles calculations based on the correlated fragment molecular orbital (FMO) indicated that two aromatic residues, Tyr224 and Phe242, form stable π-π stacking interaction with substrates. Enzyme kinetics also supported the importance of Tyr224 in catalysis: the kcat/Km value of the Y224L mutant was reduced by 300-fold than that of wild-type (WT) when utilizing either (R)-methylbenzylamine [(R)-MBA] or (R)-1-(2-naphthyl)ethylamine [(R)-NEA] as the substrate. On the other hand, several Phe242 mutants exhibited higher reactivity toward (R)-NEA than the WT enzyme. In addition, FMO analysis indicated that pkAOx forms â¼13 kcal/mol more stable interaction with (R)-MBA than with (S)-MBA; this energy difference contributes to specific recognition of (R)-MBA in the racemate. Through the present study, we clarified three features of pkAOx: the roles of Tyr224 and Phe242 in catalysis, the origin of high stereoselectivity, and the potential to extend its reactivity toward amine compounds with bulky groups.
ABSTRACT
Microfluidics is a rapidly growing field in which small volumes of liquid are moved through channels in a large variety of applications. Fabricating such channels can be expensive. Here, we describe an inexpensive method for making 3D channels in fluidic chips by using a sacrificial template made of coated metal wire or metal tubes. A 3D template is embedded in polymer or glass and then dissolved, leaving channels in the chip, without the need for expensive instruments. By changing the mold, chips of various shapes can be made.
Subject(s)
Dimethylpolysiloxanes/chemistry , Glass/chemistry , Lab-On-A-Chip Devices , Metals/chemistry , Microfluidics/instrumentation , Aluminum Oxide/chemistry , Copper/chemistry , Cost-Benefit Analysis , Equipment Design , Lab-On-A-Chip Devices/economics , Microfluidics/economics , Polyethylene Glycols/chemistry , Solubility , Steel/chemistry , Surface PropertiesABSTRACT
The deracemization of racemic amines to yield enantioenriched amines using S-stereoselective amine oxidases (AOx) has recently been attracting attention. However, R-stereoselective AOx that are suitable for deracemization have not yet been identified. An R-stereoselective AOx was now evolved from porcine kidney D-amino acid oxidase (pkDAO) and subsequently use for the deracemization of racemic amines. The engineered pkDAO, which was obtained by directed evolution, displayed a markedly changed substrate specificity towards Râ amines. The mutant enzyme exhibited a high preference towards the substrate α-methylbenzylamine and was used to synthesize the Sâ amine through deracemization. The findings of this study indicate that further investigations on the structure-activity relationship of AOx are warranted and also provide a new method for biotransformations in organic synthesis.