Spectrophotometric assay of d-isoleucine using an artificially created d-amino acid dehydrogenase

Akita, Hironaga; Imaizumi, Yoshifumi; Suzuki, Hirokazu; Doi, Kunio; Ohshima, Toshihisa

doi:10.1007/s10529-014-1597-z

Cited by 9 publications

(4 citation statements)

References 14 publications

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“…The production of ( R )‐5,5,5‐trifluoronorvaline, an intermediate for the γ‐secretase inhibitor BMS‐708163 (Figure 6), using a D ‐AADH has even been carried out on pilot plant‐scale (50 kg). Moreover, the use of an engineered D ‐AADH in a spectrophotometric assay for D ‐isoleucine has recently been reported 86…”

Section: Imine Reduction Using Defined Enzymesmentioning

confidence: 99%

Biocatalytic Imine Reduction and Reductive Amination of Ketones

2015

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Chiral aminesr epresent ap rominentf unctional group in pharmaceuticals and agrochemicals and are hence attractive targets for asymmetric synthesis.S ince the pharmaceutical industry has identified biocatalysis as av aluablet oolf or synthesising chiral molecules with high enantiomeric excess and under mild reactionc onditions,e nzymatic methods for chiral amine synthesisa re increasing in importance.A mong the strategies available in this context, the asymmetric reductiono fi mines by NAD(P)Hdependent enzymes andt he related reductive amination of ketones have long remained underrepresented. However, recent years have witnessed an impressive progress in the applicationo fn atural or engineered imine-reducing enzymes,s uch as imine reductases,o pine dehydrogenases,a mine dehydrogenases, and artificial metalloenzymes.T his review provides ac omprehensive overview of biocatalytic imine reductiona nd reductive amination of ketones,h ighlightingt he natural roles,s ubstrate scopes,s tructural features, and potential applicationf ields of the involvedenzymes.1I ntroduction 2I mine Reduction in Nature 2. 1I ntroductionChiral amines represent the core structure of am yriad of natural products as well as man-made compounds of public demand, such as pharmaceuticals and agrochemicals (Figure 1). Accordingt o recent estimates,c hiral amine moieties are present in about 40% of active pharmaceutical ingredients (APIs) and 20% of agrochemicals.[1] Moreover, optically pure amines,a mino acids,a nd amino alcohols are frequently employed in chemical synthesis as chiral auxiliariesorr esolving agents.Theirp aramount importance across several chemical disciplines andi ndustrial sectors has made chiral amines anda mino acids attractive targets for asymmetrics ynthesis.E stablished approachesf or their preparation are the stereoselective addition of nucleophiles to imines (including the famous Mannich and Strecker reactions), asymmetric C À Ha mination and hydroamination, and the asymmetric reductiono fe namines and imines,w hich may either be pre-formed or may occur as intermediates in an asymmetric reductive amination process.[2] Metallo-or organocatalytic methodsf or asymmetric imine and enamine reductiona re broadly applied, and intense research efforts are devoted to the improvement of the scope and stereoselectivity of these processes. [2,3] In cases where established asymmetric synthesism ethodsa re inapplicable or fail to provide the desired optical purity, diastereomeric salt crystallisation is still widely used for the classical resolution of racemic amines or for upgrading the enantiomeric excess of an optically enriched product.[2d]Biocatalytic transformationsa re increasingly being recognised as an attractive optionf or the asymmetric synthesiso fc hiral molecules.[4] Particularly in the

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Section: Imine Reduction Using Defined Enzymesmentioning

confidence: 99%

Biocatalytic Imine Reduction and Reductive Amination of Ketones

2015

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“…Through the use of U. thermosphaericus DAADH, we have already succeeded in developing novel methods for producing D-branched-chain amino acids (D-leucine, D-isoleucine, and D-valine) (15) (15) and for assaying D-isoleucine without interference from the three other isomers (16). As the next step, the creation of an enzyme exhibiting different specificity and higher catalytic efficiency became a key for the further development of D-amino acid production by the method at the industrial level.…”

Section: Resultsmentioning

confidence: 99%

Structure-Based Engineering of an Artificially Generated NADP ⁺ -Dependent d -Amino Acid Dehydrogenase

Hayashi

Seto

Akita

et al. 2017

Appl Environ Microbiol

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A stable NADP-dependent d-amino acid dehydrogenase (DAADH) was recently created from -diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains. In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but all include tedious steps. The use of NAD(P)-dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.

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“…This enzymatic endpoint assay consisted of two successive reaction steps ( Figure 2B ): stoichiometric NADPH generation coupled to D -isoleucine deamination catalyzed by D -AADH followed by stoichiometric formation of water-soluble formazan (WSF) from water-soluble tetrazolium-3 (WST-3) coupled to oxidation of NADPH. After the reaction conditions were optimized, D -isoleucine was determined within a range of 1.0–50 μM ( Akita et al, 2014a ). We next examined the effect of three isoleucine analogs (100 μM) on enzymatic determination of D -isoleucine (1.0–50 μM).…”

Section: Spectrophotometric Assay Of D -Isoleucinementioning

confidence: 99%

Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application

et al. 2018

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Many kinds of NAD(P)+-dependent L-amino acid dehydrogenases have been so far found and effectively used for synthesis of L-amino acids and their analogs, and for their sensing. By contrast, similar biotechnological use of D-amino acid dehydrogenase (D-AADH) has not been achieved because useful D-AADH has not been found from natural resources. Recently, using protein engineering methods, an NADP+-dependent D-AADH was created from meso-diaminopimelate dehydrogenase (meso-DAPDH). The artificially created D-AADH catalyzed the reversible NADP+-dependent oxidative deamination of D-amino acids to 2-oxo acids. The enzyme, especially thermostable one from thermophiles, was efficiently applicable to synthesis of D-branched-chain amino acids (D-BCAAs), with high yields and optical purity, and was useful for the practical synthesis of 13C- and/or 15N-labeled D-BCAAs. The enzyme also made it possible to assay D-isoleucine selectively in a mixture of isoleucine isomers. Analyses of the three-dimensional structures of meso-DAPDH and D-AADH, and designed mutations based on the information obtained made it possible to markedly enhance enzyme activity and to create D-AADH homologs with desired reactivity profiles. The methods described here may be an effective approach to artificial creation of biotechnologically useful enzymes.

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Spectrophotometric assay of d-isoleucine using an artificially created d-amino acid dehydrogenase

Cited by 9 publications

References 14 publications

Biocatalytic Imine Reduction and Reductive Amination of Ketones

Biocatalytic Imine Reduction and Reductive Amination of Ketones

Structure-Based Engineering of an Artificially Generated NADP ⁺ -Dependent d -Amino Acid Dehydrogenase

Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application

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Spectrophotometric assay of d-isoleucine using an artificially created d-amino acid dehydrogenase

Cited by 9 publications

References 14 publications

Biocatalytic Imine Reduction and Reductive Amination of Ketones

Biocatalytic Imine Reduction and Reductive Amination of Ketones

Structure-Based Engineering of an Artificially Generated NADP + -Dependent d -Amino Acid Dehydrogenase

Artificial Thermostable D-Amino Acid Dehydrogenase: Creation and Application

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Product

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Structure-Based Engineering of an Artificially Generated NADP ⁺ -Dependent d -Amino Acid Dehydrogenase