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The high-valent iron-oxo species formed in the non-heme diiron enzymes have high oxidative reactivity and catalyze difficult chemical reactions. Although the hydroxylation of inert methyl groups is an industrially promising reaction, utilizing non-heme diiron enzymes as such a biocatalyst has been difficult. Here we show a three-component monooxygenase system for the selective terminal hydroxylation of α-aminoisobutyric acid (Aib) into α-methyl-D-serine. It consists of the hydroxylase component, AibH1H2, and the electron transfer component. Aib hydroxylation is the initial step of Aib catabolism in Rhodococcus wratislaviensis C31-06, which has been fully elucidated through a proteome analysis. The crystal structure analysis revealed that AibH1H2 forms a heterotetramer of two amidohydrolase superfamily proteins, of which AibHm2 is a non-heme diiron protein and functions as a catalytic subunit. The Aib monooxygenase was demonstrated to be a promising biocatalyst that is suitable for bioprocesses in which the inert C–H bond in methyl groups need to be activated. Makoto Hibi et al. report a novel three-component monooxygenase system in Rhodococcus wratislaviensis. This enzyme catalyzes the activation of an inert C–H bond and may be potentially important as a biocatalyst for industrial applications. [ABSTRACT FROM AUTHOR]

Maltol derivatives are used in a variety of fields due to their metal-chelating abilities. In the previous study, it was found that cytochrome P450 monooxygenase, P450nov, which has the ability to effectively convert the 2-methyl group in a maltol derivative, transformed 3-benzyloxy-2-methyl-4-pyrone (BMAL) to 2-(hydroxymethyl)-3-(phenylmethoxy)-4H-pyran-4-one (BMAL-OH) and slightly to 3-benzyloxy-4-oxo-4 H-pyran-2-carboxaldehyde (BMAL-CHO). We isolated Pseudomonas nitroreducens SB32154 with the ability to convert BMAL-CHO to BMAL-COOH from soil. The enzyme responsible for aldehyde oxidation, a BMAL-CHO dehydrogenase, was purified from P. nitroreducens SB32154 and characterized. The purified BMAL-CHO dehydrogenase was found to be a xanthine oxidase family enzyme with unique structure of heterodimer composed of 75 and 15 kDa subunits containing a molybdenum cofactor and [Fe-S] clusters, respectively. The enzyme showed broad substrate specificity toward benzaldehyde derivatives. Furthermore, one-pot conversion of BMAL to BMAL-COOH via BMAL-CHO by the combination of the BMAL-CHO dehydrogenase with P450nov was achieved. Enzymatic conversion of BMAL to BMAL-COOH by P450nov from Novosphingobium sp. SB32149 and BMAL-CHO dehydrogenase from Pseudomonas nitroreducens SB32154 [ABSTRACT FROM AUTHOR]

Microorganisms were screened for transribosylation activity between 2′- O -methyluridine (2′-OMe-UR) and nucleobases, for the purpose of developing a biotransformation process to synthesize 2′- O -methylribonucleosides (2′-OMe-NRs), which are raw materials for nucleic acid drugs. An actinomycete, Agromyces sp. MM-1 was found to produce 2′- O -methyladenosine (2′-OMe-AR) when whole cells were used in a reaction mixture containing 2′-OMe-UR and adenine. The enzyme responsible for the transribosylation was partially purified from Agromyces sp. MM-1 cells through a six-step separation procedure, and identified as a nucleoside hydrolase family enzyme termed Ag NH. Ag NH was a bi-functional enzyme catalyzing both hydrolysis towards 2′-OMe-NRs and transribosylation between 2′-OMe-UR and various nucleobases as well as adenine. In the hydrolysis reaction, Ag NH preferred guanosine analogues as its substrates. In the transribosylation reaction, Ag NH showed strong activity towards 6-chloroguanine, with 25-fold relative activity when adenine was used as the acceptor substrate. The transribosylation reaction product from 2′-OMe-UR and 6-chloroguanine was determined to 2′- O -methyl-6-chloroguanosine (2′-OMe-6ClGR). Under the optimal conditions, the maximum molar yield of 2′-OMe-6ClGR reached 2.3% in a 293-h reaction, corresponding to 440 mg/L. [ABSTRACT FROM AUTHOR]

2′- O -Methylribonucleosides (2′-OMe-NRs) are promising raw materials for the production of nucleic acid drugs. We previously reported that Lb NH, a nucleoside hydrolase from Lactobacillus buchneri LBK78 (NITE P-01581), was the first enzyme found to act on 2′-OMe-NRs. In the present study, we determined that Lb NH also has the transribosylation activity between 2′-OMe-NRs and nucleobases, in addition to the hydrolyzing activity towards 2′-OMe-NRs. When 2′- O -methyluridine (2′-OMe-UR) and adenine were reacted with Lb NH, 2′- O -methyladenosine (2′-OMe-AR) was produced. Lb NH preferred purine nucleobases as its acceptor substrates for the transribosylation with 2′-OMe-UR as a donor substrate. Kinetic analysis of Lb NH revealed that adenine behaved as a mixed inhibitor of the hydrolysis of 2′-OMe-UR. Under the optimal reaction conditions, the maximum molar yield of enzymatic 2′-OMe-AR produced reached 0.97% towards 2′-OMe-UR, corresponding to 0.16 g/L. [ABSTRACT FROM AUTHOR]

The recent use of optically active 3-substituted gamma-aminobutyric acid (GABA) analogs in human therapeutics has identified a need for an efficient, stereoselective method of their synthesis. Here, bacterial strains were screened for enzymes capable of stereospecific hydrolysis of 3-substituted glutarimides to generate ( R)-3-substituted glutaric acid monoamides. The bacteria Alcaligenes faecalis NBRC13111 and Burkholderia phytofirmans DSM17436 were discovered to hydrolyze 3-(4-chlorophenyl) glutarimide (CGI) to ( R)-3-(4-chlorophenyl) glutaric acid monoamide (CGM) with 98.1 % enantiomeric excess (e.e.) and 97.5 % e.e., respectively. B. phytofirmans DSM17436 could also hydrolyze 3-isobutyl glutarimide (IBI) to produce ( R)-3-isobutyl glutaric acid monoamide (IBM) with 94.9 % e.e. BpIH, an imidase, was purified from B. phytofirmans DSM17436 and found to generate ( R)-CGM from CGI with specific activity of 0.95 U/mg. The amino acid sequence of BpIH had a 75 % sequence identity to that of allantoinase from A. faecalis NBRC13111 (AfIH). The purified recombinant BpIH and AfIH catalyzed ( R)-selective hydrolysis of CGI and IBI. In addition, a preliminary investigation of the enzymatic properties of BpIH and AfIH revealed that both enzymes were stable in the range of pH 6-10, with an optimal pH of 9.0, stable at temperatures below 40 °C, and were not metalloproteins. These results indicate that the use of this class of hydrolase to generate optically active 3-substituted glutaric acid monoamide could simplify the production of specific chiral GABA analogs for drug therapeutics. [ABSTRACT FROM AUTHOR]

A novel enzymatic production system of optically pure β-hydroxy α-amino acids was developed. Two enzymes were used for the system: an N-succinyl L-amino acid β-hydroxylase (SadA) belonging to the iron(II)/α-ketoglutarate-dependent dioxygenase superfamily and an N-succinyl L-amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N-succinyl aliphatic L-amino acids and produced N-succinyl β-hydroxy L-amino acids, such as N-succinyl- L-β-hydroxyvaline, N-succinyl- L-threonine, (2 S,3 R)- N-succinyl- L-β-hydroxyisoleucine, and N-succinyl- L- threo-β-hydroxyleucine. LasA catalyzed the desuccinylation of various N-succinyl- L-amino acids. Surprisingly, LasA is the first amide bond-forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L-leucine. By combining SadA and LasA in a preparative scale production using N-succinyl- L-leucine as substrate, 2.3 mmol of L- threo-β-hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β-hydroxy α-amino acids. [ABSTRACT FROM AUTHOR]

The asymmetric hydroxylation of inactive carbon atoms is still an important reaction in the industrial synthesis of valuable chiral compounds such as pharmaceuticals and fine chemicals. Applications of monooxygenation enzymes, like cytochrome P450 monooxygenases, flavin-containing monooxygenases, and Fe(II)/α-ketoglutarate-dependent dioxygenases (Fe/αKG-DOs), are strongly desired as hydroxylation biocatalysts because they have great advantages in regio- and stereoselectivity of the reactions. Recently, several novel Fe/αKG-DOs have been found to catalyze the asymmetric hydroxylation of aliphatic amino acids. Depending on their amino acid sequences, these Fe/αKG-DOs catalyze different types of regioselective hydroxylations, or C3-, C4-, and C5-hydroxylation. Additionally, most also have stereoselective sulfoxidation activities. Here, we have reviewed the characterization and process development of this novel functioning group of Fe/αKG-DOs. [ABSTRACT FROM AUTHOR]

Abstract: Asymmetric sulfoxidation of sulfur-containing l-amino acids was successfully achieved through bioconversion using IDO, which is an Fe(II)/α-ketoglutarate-dependent dioxygenase previously found in Bacillus thuringiensis strain 2e2. The IDO catalyzed sulfoxidation of l-methionine, l-ethionine, S-methyl-l-cysteine, S-ethyl-l-cysteine, and S-allyl-l-cysteine into the corresponding (S)-configured sulfoxides such as (+)-methiin and (+)-alliin, which are responsible for valuable physiological activities in mammals, and have high stereoselectivity. Herein we have established an effective preparative laboratory scale production method to obtain enantiomerically pure chiral sulfoxides using an IDO biocatalyst. [Copyright &y& Elsevier]

l-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of l-leucine and l-norleucine into (2 S,4 S)-5-hydroxyleucine and (2 S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of l-methionine and l-ethionine in the same manner as previously described l-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids. [ABSTRACT FROM AUTHOR]

We determined the enzymatic characteristics of an industrially important biocatalyst, a-ketoglutarate-dependent L-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic L-amino acids, as well as L-isoleucine, and produced (S)-3-hydroxy-L-allo-isoleucine, 4-hydroxy-L-leucine, (S)-4-hydroxy-L-norvaline, 4-hydroxy-L-norleucine, and 5-hydroxy-L-norleucine. The IDO reaction product of L-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing L-amino acids and generated L-methionine sulfoxide and L-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst. [ABSTRACT FROM AUTHOR]

Three extracellular ligninolytic oxidoreductases that are produced by a commercial laccase-producing Trametes sp. Ha1 were purified and characterized. This fungus showed strong ligninolytic oxidoreductase activity with and without hydrogen peroxide present in the reaction mixture. The oxidoreductase activity was found to be derived from two laccases and a peroxidase. One of the two laccases represents a main component of the commercial laccase preparation from Trametes sp. Ha1. This enzyme had a high thermostability, which makes it attractive for practical applications. The second laccase was induced by the addition of p-xylidine into the culture medium and showed unique characteristics with respect to pI value and substrate specificity. The peroxidase showed wide oxidation activity against aromatic compounds. [Copyright &y& Elsevier]

Transcriptome data for a xylitol-producing recombinant Escherichia coli were obtained and used to tune up its productivity. Structural genes of NADPH-dependent D-xyloSe reductase and D-xylose permease were inserted into an Escherichia coli chromosome to construct a recombinant strain producing xylitol from n-xylose for use as a model system for NADPH-dependent bioconversion. Transcriptome analysis of xylitol-producing and nonproducing conditions for the recombinant revealed that xylitol production down-regulated 56 genes. These genes were then selected as candidate factors for suppression of the NADPH supply and were disrupted to validate their functions. Of the gene disruptants, that resulting from the deletion of yhbC showed the best bioconversion rate. Also, the deletion accelerated cell growth during log phase. The features of the mutant could be maintained in jar fermenter-scale production of xylitol. Thus, our novel molecular host strain breeding method using transcriptome analysis was fully effective and could be applied to improving various industrial strains. [ABSTRACT FROM AUTHOR]

Abstract: Pseudomonas putida vanillate-O-demethylase consisting of VanA and VanB was expressed in Escherichia coli strain K-12. Recombinant E. coli strain K-12 cells expressing VanAB efficiently converted vanillate into protocatechuate with glucose consumption. Mutant lacking either pgi or zwf showed higher or lower converting activity than the parental strain, respectively. Formaldehyde, which is the by-product of the demethylation, was converted into formate in the cellular reaction. Formate accumulation was blocked by gene disruption of the E. coli frmA that coded glutathione-dependent formaldehyde dehydrogenase. [Copyright &y& Elsevier]

Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP+ or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD+, NADP+, flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors. • Uracil-thymine dehydrogenase was found to be a heteromer consisting of three different subunits. • Uracil-thymine dehydrogenase catalyzed oxidation of uracil with artificial electron acceptors. • The activity of uracil-thymine dehydrogenase was enhanced by cerium ions. [ABSTRACT FROM AUTHOR]

The front cover picture, provided by Makoto Hibi, Jun Ogawa et al., depicts Fe(II)/α ‐ ketoglutarate (αKG) ‐ dependent dioxygenases, which are hydroxylation biocatalysts catalyzing highly selective monooxygenation available to industrial applications. Recently, a great number of Fe(II)/αKG ‐ dependent dioxygenases with a variety of functions has been found in the genome database, and collected into a biocatalyst library. One of these enzymes, N ‐ succinyl L ‐ amino acid β ‐ hydroxylase, was applied to an enzymatic cascade system together with N ‐ succinyl L ‐ amino acid desuccinylase for the production of optically pure β ‐ hydroxy α ‐ amino acids. Details can be found in the full paper on pages 767 – 774 (M. Hibi, T. Kasahara, T. Kawashima, H. Yajima, S. Kozono, S. V. Smirnov, T. Kodera, M. Sugiyama, S. Shimizu, K. Yokozeki, and J. Ogawa, Adv. Synth. Catal. 2015, 357, 767 – 774; DOI: 10.1002/adsc.201400672). [ABSTRACT FROM AUTHOR]

A wide variety of S-substituted cysteine derivatives occur in plant metabolites. For example, S -allyl- l -cysteine (SAC), mainly contained in garlic, gathers huge interest because of its favorable bioactivities for human health. However, conventional methods for preparing SAC suffer from several drawbacks with regard to efficiency and toxicity, which highlights the need for improved processes for SAC synthesis. This study aims to develop a novel bioprocess to produce SAC by microbial enzymes from easily available substrates. We found that Escherichia coli had the ability to synthesize SAC from allyl mercaptan, pyruvic acid, and ammonium sulfate. An enzyme purification through 3-step column chromatography, followed by determination of the N-terminal amino acid sequence revealed that tryptophanase (TnaA) was the enzyme responsible for SAC formation. Although the enzyme catalyzed the reversible reaction for synthesizing and degrading SAC, the degradation proceeded significantly faster than the synthesis. Interestingly, TnaA catalyzed the synthesis of a wide range of S-substituted cysteines with alkyl chains or aromatic rings, some of which are present in Allium and Petiveria plants. Our results showed a novel substrate specificity of TnaA toward various S-substituted cysteine. TnaA is a promising biocatalyst for developing a new process to supply various valuable S-substituted cysteine derivatives for medicinal and health-promoting applications. [ABSTRACT FROM AUTHOR]

Maltol derivatives are utilized in a variety of fields due to their metal-chelating abilities, and modification of the 2-methyl side chain is known to effectively expand their functional diversity. In the present study, microbial enzymes were screened for hydroxylating activity towards the 2-methyl group in a maltol derivative, 3-benzyloxy-2-methyl-4-pyrone (BMAL). Novosphingobium sp. SB32149 was found to have the ability to convert BMAL into 3-benzyloxy-2-hydroxymethyl-4-pyrone (BMAL-OH). The enzymes responsible, a cytochrome P450 monooxygenase (P450nov), a ferredoxin (FDXnov), and a ferredoxin reductase (FDRnov), were identified in the SB32149 strain. In the reaction with recombinant Escherichia coli expressing P450nov, FDXnov, and FDRnov, BMAL-OH was successfully produced from BMAL. Moreover, using the directed evolution approach, four amino acid substitutions, L188P/F218L/L237M in P450nov and A10T in FDXnov, were found to enhance BMAL-OH production. Consequently, up to 5.2 g/L BMAL-OH was obtained from 8.0 g/L BMAL by bioconversion using a 250-mL jar fermenter, indicating that this strain may be useful for synthesis of maltol derivatives which could have potential applications in various fields. [ABSTRACT FROM AUTHOR]

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids inLactobacillus plantarumas substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids. Oxidative cleavage of oxo unsaturated fatty acid into dicarboxylic acid by laccase. [ABSTRACT FROM PUBLISHER]

2′-O-Methylribonucleosides (2′-OMe-NRs) are promising raw materials for nucleic acid drugs because of their high thermal stability and nuclease tolerance. In the course of microbial screening for metabolic activity toward 2′-OMe-NRs,Lactobacillus buchneriLBK78 was found to decompose 2′-O-methyluridine (2′-OMe-UR). The enzyme responsible was partially purified fromL. buchneriLBK78 cells by a four-step purification procedure, and identified as a novel nucleoside hydrolase. This enzyme,LbNH, belongs to the nucleoside hydrolase superfamily, and formed a homotetrameric structure composed of subunits with a molecular mass around 34 kDa.LbNH hydrolyzed 2′-OMe-UR to 2′-O-methylribose and uracil, and the kinetic constants wereKmof 0.040 mM,kcatof 0.49 s−1, andkcat/Kmof 12 mM−1 s−1. In a substrate specificity analysis,LbNH preferred ribonucleosides and 2′-OMe-NRs as its hydrolytic substrates, but reacted weakly with 2′-deoxyribonucleosides. In a phylogenetic analysis,LbNH showed a close relationship with purine-specific nucleoside hydrolases from trypanosomes. LbNH, a novel nucleoside hydrolase fromLactobacillus buchneriLBK 78 catalyzes 1'-hydrolysis reaction toward 2'-O-methyluridine to form 2'-O-methylribose and uracil as the degradation products. [ABSTRACT FROM PUBLISHER]

Trehalose confers protection against various environmental stresses on yeast cells. In this study, trehalase gene deletion mutants that accumulate trehalose at high levels showed significant stress tolerance to acetic acid. The enhancement of trehalose accumulation can thus be considered a target in the breeding of acetic acid-tolerant yeast strains. [ABSTRACT FROM AUTHOR]

All life generates deoxyribonucleotides, the building blocks of DNA, via ribonucleotide reductases (RNRs). The complexity of this reaction suggests it did not evolve until well after the advent of templated protein synthesis, which in turn suggests DNA evolved later than both RNA and templated protein synthesis. However, deoxyribonucleotides may have first been synthesised via an alternative, chemically simpler route-the reversal of the deoxyriboaldolase (DERA) step in deoxyribonucleotide salvage. In light of recent work demonstrating that this reaction can drive synthesis of deoxyribonucleosides, we consider what pressures early adoption of this pathway would have placed on cell metabolism. This in turn provides a rationale for the replacement of DERA-dependent DNA production by RNR-dependent production. [ABSTRACT FROM AUTHOR]

l - threo -3,4-Dihydroxyphenylserine ( l -DOPS, Droxidopa) is a psychoactive drug and synthetic amino acid precursor that acts as a prodrug to the neurotransmitters. SadA, a dioxygenase from Burkholderia ambifaria AMMD, is an Fe(II)- and α-ketoglutarate (KG)-dependent enzyme that catalyzes N -substituted branched-chain or aromatic l -amino acids. SadA is able to produce N -succinyl- l - threo -3,4-dimethoxyphenylserine (NSDOPS), which is a precursor of l -DOPS, by catalyzing the hydroxylation of N -succinyl-3,4-dimethoxyphenylalanine (NSDOPA). However, the catalytic activity of SadA toward NSDOPS is much lower than that toward N -succinyl branched-chain l -amino acids. Here, we report an improved biocatalytic synthesis of NSDOPS with SadA. Structure-based protein engineering was applied to improve the α-KG turnover activity for the synthesis of NSDOPS. The G79A, G79A/F261W or G79A/F261R mutant showed a more than 6-fold increase in activity compared to that of the wild-type enzyme. The results provide a new insight into the substrate specificity toward NSDOPA and will be useful for the rational design of SadA mutants as a target of industrial biocatalysts. [ABSTRACT FROM AUTHOR]

An enzyme exhibiting oxidase activity for β-lactoglobulin, myoglobin, and l-lysine-containing peptides was found from a newly isolated fungal strain, Penicillium steckii AIU 027. The enzyme also oxidized l-amino acids, N α-benzyloxycarbonyl-l-lysine (N α-Z-l-lysine) and N ε-Z-l-lysine, but not d-amino acids and amines. Thus, the enzyme was classified into a group of l-amino acid oxidases (l-AAOs). However, characteristics of this l-AAO were significantly different from those of other l-AAOs as follows. The l-AAO from P. steckii AIU 027 oxidized both the α-amino group and the ε-amino group in l-amino acids and l-lysine-containing peptides, and the K m values for l-lysine-containing polypeptides were lower than those for N α-Z-l-lysine and l-lysine-containing dipeptides. The enzyme contained flavin and iron, and composed of four identical subunits with molecular mass of 75.3 kDa. The N-terminal amino acid sequence, ENIADVADAMGPWFDGVAYMKSKKN, was different from that of other l-AAOs. Thus, the l-AAO with protein oxidase activity was first reported here from P. steckii AIU 027. [Copyright &y& Elsevier]

An l-specific amino acid oxidase (l-AAO) suitable for assay of N-acyl-l-amino acid amidohydrolase (l-aminoacylase) activity was purified from Rhodococcus sp. AIU LAB-3. The enzyme exhibited broad substrate specificity and catalyzed an oxidative deamination of the α-amino group of l-amino acids. The optimal enzyme activities for l-amino acids tested were observed in the pH range from 6.0 to 8.5, and more than 80% of the maximum activity was obtained at pH 7.5. The enzyme was stable in the pH range from 7.0 to 8.5, and the apparent K m values for those l-amino acids were small. We, therefore, developed a new enzymatic method for assay of l-aminoacylase activity using the l-AAO at pH 7.5. The new enzymatic method had advantages that the l-aminoacylase reaction was spectrophotometrically followed by measuring absorbance at 555 nm. The l-aminoacylase activity was assayed within 10 min using a small reaction volume. Thus, the new enzymatic method was simple and sensitive compared to the ninhydrin method. [Copyright &y& Elsevier]

A novel dioxygenase from Burkholderia ambifaria AMMD (SadA) stereoselectively catalyzes the C3-hydroxylation of N-substituted branched-chain or aromatic L-amino acids, especially N-succinyl-L-leucine, coupled with the conversion of α-ketoglutarate to succinate and CO2. To elucidate the structural basis of the substrate specificity and stereoselective hydroxylation, we determined the crystal structures of the SadA.Zn(II) and SadA.Zn(II).α-KG complexes at 1.77 Å and 1.98 Å resolutions, respectively. SadA adopted a double-stranded β-helix fold at the core of the structure. In addition, an HXD/EXnH motif in the active site coordinated a Zn(II) as a substitute for Fe(II). The α-KG molecule also coordinated Zn(II) in a bidentate manner via its 1-carboxylate and 2-oxo groups. Based on the SadA.Zn(II).α-KG structure and mutation analyses, we constructed substrate-binding models with N-succinyl-L-leucine and N-succinyl-L-phenylalanine, which provided new insight into the substrate specificity. The results will be useful for the rational design of SadA variants aimed at the recognition of various N-succinyl L-amino acids. [ABSTRACT FROM AUTHOR]

l-isoleucine-4-hydroxylase ( IDO) is a recently discovered member of the Pfam family PF10014 (the former DUF 2257 family) of uncharacterized conserved bacterial proteins. To uncover the range of biochemical activities carried out by PF10014 members, eight in silico-selected IDO homologues belonging to the PF10014 were cloned and expressed in Escherichia coli. l-methionine, l-leucine, l-isoleucine and l-threonine were found to be catalysed by the investigated enzymes, producing l-methionine sulfoxide, 4-hydroxyleucine, 4-hydroxyisoleucine and 4-hydroxythreonine, respectively. An investigation of enzyme kinetics suggested the existence of a novel subfamily of bacterial dioxygenases within the PF10014 family for which free l-amino acids could be accepted as in vivo substrates. A hypothesis regarding the physiological significance of hydroxylated l-amino acids is also discussed. [ABSTRACT FROM AUTHOR]

Background: Reproduction and sustainability are important for future society, and bioprocesses are one technology that can be used to realize these concepts. However, there is still limited variation in bioprocesses and there are several challenges, especially in the operation of energy-requiring bioprocesses. As an example of a microbial platform for an energy-requiring bioprocess, we established a process that efficiently and enzymatically synthesizes 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase. This method consists of the coupling reactions of the reversible nucleoside degradation pathway and energy generation through the yeast glycolytic pathway. Results: Using E. coli that co-express deoxyriboaldolase and phosphopentomutase, a high amount of 2′-deoxyribonucleoside was produced with efficient energy transfer under phosphate-limiting reaction conditions. Keeping the nucleobase concentration low and the mixture at a low reaction temperature increased the yield of 2′-deoxyribonucleoside relative to the amount of added nucleobase, indicating that energy was efficiently generated from glucose via the yeast glycolytic pathway under these reaction conditions. Using a one-pot reaction in which small amounts of adenine, adenosine, and acetone-dried yeast were fed into the reaction, 75 mM of 2′-deoxyinosine, the deaminated product of 2′-deoxyadenosine, was produced from glucose (600 mM), acetaldehyde (250 mM), adenine (70 mM), and adenosine (20 mM) with a high yield relative to the total base moiety input (83%). Moreover, a variety of natural dNSs were further synthesized by introducing a base-exchange reaction into the process. Conclusion: A critical common issue in energy-requiring bioprocess is fine control of phosphate concentration. We tried to resolve this problem, and provide the convenient recipe for establishment of energy-requiring bioprocesses. It is anticipated that the commercial demand for dNSs, which are primary metabolites that accumulate at very low levels in the metabolic pool, will grow. The development of an efficient production method for these compounds will have a great impact in both fields of applied microbiology and industry and will also serve as a good example of a microbial platform for energy-requiring bioprocesses. [ABSTRACT FROM AUTHOR]

4-Hydroxyisoleucine (HIL) found in fenugreek seeds has insulinotropic and anti-obesity effects and is expected to be a novel orally active drug for insulin-independent diabetes. Here, we show that the newly isolated strain Bacillus thuringiensis 2e2 and the closely related strain B. thuringiensis ATCC 35646 operate a novel metabolic pathway for l-isoleucine ( l-Ile) via HIL and 2-amino-3-methyl-4-ketopentanoic acid (AMKP). The HIL synthesis was catalyzed stereoselectively by an α-ketoglutaric acid-dependent dioxygenase and to be useful for efficient production of a naturally occurring HIL isomer, (2 S,3 R,4 S)-HIL. The (2 S,3 R,4 S)-HIL was oxidized to (2 S,3 R) -AMKP by a NAD-dependent dehydrogenase. The metabolic pathway functions as an effective bypass pathway that compensates for the incomplete tricarboxylic acid (TCA) cycle in Bacillus species and also explains how AMKP, a vitamin B antimetabolite with antibiotic activity, is synthesized. These novel findings pave a new way for the commercial production of HIL and also for AMKP. [ABSTRACT FROM AUTHOR]

The stereo-specific l-isoleucine-4-hydroxylase ( l-isoleucine dioxygenase (IDO)) was cloned and expressed in an Escherichia coli 2Δ strain lacking the activities of α-ketoglutarate dehydrogenase (EC 1.2.4.2), isocitrate liase (EC 4.1.3.1), and isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5). The 2Δ strain could not grow in a minimal-salt/glucose/glycerol medium due to the blockage of TCA during succinate synthesis. The IDO activity in the 2Δ strain was able to “shunt” destroyed TCA, thereby coupling l-isoleucine hydroxylation and cell growth. Using this strain, we performed the direct biotransformation of l-isoleucine into 4-HIL with an 82% yield. [ABSTRACT FROM AUTHOR]

Abstract: The unique function of 4-hydroxyisoleucine (4-HIL) is to stimulate glucose-induced insulin secretion in a glucose-dependent manner. 4-HIL is distributed only in certain kinds of plants and mushrooms, but the biosynthetic mechanism of 4-HIL has not been elucidated. Moreover, 4-HIL-producing microorganisms have not been reported. l-isoleucine (l-Ile) hydroxylating activity producing 4-HIL was detected in a cell lysate of Bacillus thuringiensis strain 2e2 AKU 0251 obtained from the mid-late exponential phase of growth. Properties of the purified hydroxylase demonstrated that it is a α-ketoglutaric acid (α-KG) dependent l-Ile dioxygenase (IDO) and requires α-KG, ferric ion, and ascorbic acid for its maximum activity. IDO showed high stereoselectivity in l-Ile hydroxylation producing only (2S,3R,4S)-4-HIL. The N-terminal 22 amino acids sequence revealed high homology to a hypothetical protein (GenBank ID: RBTH_06809) in B. thuringiensis serovar israelensis ATCC 35646. The histidine motif, which is conserved in α-KG dependent dioxygenases, is found in RBTH_06809. [Copyright &y& Elsevier]