Cloning and characterization of decaprenyl diphosphate synthase from three different fungi.

Coenzyme Q (CoQ) is composed of a benzoquinone moiety and an isoprenoid side chain of varying lengths. The length of the side chain is controlled by polyprenyl diphosphate synthase. In this study, dps1 genes encoding decaprenyl diphosphate synthase were cloned from three fungi: Bulleromyces albus, Saitoella complicata, and Rhodotorula minuta. The predicted Dps1 proteins contained seven conserved domains found in typical polyprenyl diphosphate synthases and were 528, 440, and 537 amino acids in length in B. albus, S. complicata, and R. minuta, respectively. Escherichia coli expressing the fungal dps1 genes produced CoQ10 in addition to endogenous CoQ8. Two of the three fungal dps1 genes (from S. complicata and R. minuta) were able to replace the function of ispB in an E. coli mutant strain. In vitro enzymatic activities were also detected in recombinant strains. The three dps1 genes were able to complement a Schizosaccharomyces pombe dps1, dlp1 double mutant. Recombinant S. pombe produced mainly CoQ10, indicating that the introduced genes were independently functional and did not require dlp1. The cloning of dps1 genes from various fungi has the potential to enhance production of CoQ10 in other organisms.

Efficient synthesis of (R)-3-hydroxypentanenitrile in high enantiomeric excess by enzymatic reduction of 3-oxopentanenitrile.

(R)-3-Hydroxypentanenitrile (HPN) is an important intermediate in the synthesis of an immunosuppressive inosine 5'-monophosphate dehydrogenase inhibitor. An efficient enzymatic procedure for the synthesis of (R)-HPN with over 99 % enantiomeric excess using a novel acetoacetyl-CoA reductase (AdKR) from Achromobacter denitrificans was successfully established. Many microorganisms are known to reduce 3-oxopentannitrile (KPN) to (R)-HPN. An enzyme from A. denitrificans partially purified using ion exchange chromatography reduced KPN to (R)-HPN with high enantioselectivity. The AdKR gene was cloned and sequenced and found to comprise 738 bp and encode a polypeptide of 26,399 Da. The deduced amino acid sequence showed a high degree of similarity to those of other putative acetoacetyl-CoA reductases and putative 3-ketoacyl-ACP reductases. The AdKR gene was singly expressed and coexpressed together with a glucose dehydrogenase (GDH) as a coenzyme regenerator in Escherichia coli under the control of the lac promoter. (R)-HPN was synthesized with over 99 % e.e. using a cell-free extract of recombinant E. coli cells coexpressing AdKR and GDH.

Efficient preparation of (R)-3-hydroxypentanenitrile with high enantiomeric excess by enzymatic reduction with subsequent enhancement of the optical purity by lipase-catalyzed ester hydrolysis.

An efficient chemo-enzymatic procedure for the synthesis of (R)-3-hydroxypentanenitrile (1) with over 99% enantiomeric excess using two enzymatic reactions was successfully established. Initial enantioselective enzymatic reduction of 3-oxopentanenitrile with reductase S1 gave (R)-1 with an 81.5% ee which was then converted to (R)-1-(cyanomethyl) propyl n-butyrate (3b). Subsequent lipase-catalyzed enantioselective hydrolysis of 3b gave (R)-1 in a high yield with over 99% ee.

Cloning and sequencing of a gene encoding of aldehyde oxidase in Pseudomonas sp. AIU 362.

We have cloned a gene encoding an aldehyde oxidase (ALOD) oxidized glyoxal but not glyoxylic acid from Pseudomonas sp. AIU 362. The ALOD gene contained an open reading frame consisting of 888 nucleotides corresponding to 295 amino acid residues. The deduced amino acid sequence exhibited a high similarity to those of 3-hydroxyisobutyrate dehydrogenases (3-HIBDHs). We expressed the cloned gene as an active product in Escherichia coli BL21 cells. The productivity (total units per culture broth volume) of the recombinant ALOD expressed in E. coli BL21 was 20,000-fold higher than that of ALOD in Pseudomonas sp. AIU 362. The recombinant ALOD exhibited ALOD activity and 3-HIBDH activity. The 3-HIBDH from Pseudomonas putida KT2440 also exhibited ALOD activity. Thus, the ALOD from Pseudomonas sp. AIU 362 and 3-HIBDH from P. putida KT2440 were classified into the same enzyme group.

A novel transaminase, (R)-amine:pyruvate aminotransferase, from Arthrobacter sp. KNK168 (FERM BP-5228): purification, characterization, and gene cloning.

A novel (R)-amine transaminase, which catalyzed (R)-enantioselective transamination of chiral amine, was purified to homogeneity from Arthrobacter sp. KNK168 (FERM BP-5228). The molecular mass of the enzyme was estimated to be 148 kDa by gel filtration and 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting a homotetrameric structure. The enzyme catalyzed transamination between amines and pyruvate stereo-specifically. The reaction on 1-methylbenzylamine was (R)-enantioselective. Pyruvate was the best amino acceptor, but the enzyme showed broad amino acceptor specificity for various ketone and aldehyde compounds. The apparent K(m)s for (R)-1-methylbenzylamine and pyruvate were 2.62 and 2.29 mM, respectively. The cloned gene of the enzyme consists of an open reading frame (ORF) of 993 bp encoding a protein of 330 amino acids, with a calculated molecular weight of 36,288. The deduced amino acid sequence was found to be homologous to those of the aminotransferases belonging to fold class IV of pyridoxal-5'-phosphate-dependent enzymes, such as branched-chain amino acid aminotransferases.

Microbial oxidases catalyzing conversion of glycolaldehyde into glyoxal.

The present paper reviews oxidases catalyzing conversion of glycolaldehyde into glyoxal. The enzymatic oxidation of glycolaldehyde into glyoxal was first reported in alcohol oxidases (AODs) from methylotrophic yeasts such as Candida and Pichia, and glycerol oxidase (GLOD) from Aspergillus japonicus, although it had been reported that these enzymes are specific to short-chain linear aliphatic alcohols and glycerol, respectively. These enzymes continuously oxidized ethylene glycol into glyoxal via glycolaldehyde. The AODs produced by Aspergillus ochraceus and Penicillium purpurescens also oxidized glycolaldehyde. A new enzyme exhibiting oxidase activity for glycolaldehyde was reported from a newly isolated bacterium, Paenibacillus sp. AIU 311. The Paenibacillus enzyme exhibited high activity for aldehyde alcohols such as glycolaldehyde and glyceraldehyde, but not for methanol, ethanol, ethylene glycol or glycerol. The deduced amino acid sequence of the Paenibacillus AOD was similar to that of superoxide dismutases (SODs), but not to that of methylotrophic yeast AODs. Then, it was demonstrated that SODs had oxidase activity for aldehyde alcohols including glycolaldehyde. The present paper describes characteristics of glycolaldehyde oxidation by those enzymes produced by different microorganisms.

Purification and characterization of a novel (S)-enantioselective transaminase from Pseudomonas fluorescens KNK08-18 for the synthesis of optically active amines.

Pseudomonas fluorescens KNK08-18, showing (S)-selective transaminase activity, was isolated from soil by an enrichment culture method using (S)-7-methoxy-2-aminotetraline as the main nitrogen source. A transaminase was purified from the strain to homogeneity in seven steps. The relative mass of the enzyme was estimated to be 53 kDa on SDS-polyacrylamide gel electrophoresis and 120 kDa by gel filtration, suggesting a homodimeric structure. The optimal pH and temperature for enzyme activity were about 8.0-8.5 and 40 °C. The purified enzyme produced (S)-7-methoxy-2-aminotetraline, (S)-SMA, from 7-methoxy-2-tetralone (SMT) with high enantioselectivity. Although (S)-1-phenylethylamine was the best amino donor, ß-alanine and 4-aminobutyric acid, which are good substrates for typical ω-amino acid transaminase (EC 2.6.1.18) and GABA transaminase (2.6.1.19), were not reacted. It aminated a broad range of carbonyl compounds containing aromatic, non-aromatic, and acidic and non-acidic substrates.

Cloning and overexpression of an NADH-dependent alcohol dehydrogenase gene from Candida maris involved in (R)-selective reduction of 5-acetylfuro[2,3-c]pyridine.

5-((R)-1-Hydroxyethyl)-furo[2,3-c]pyridine ((R)-FPH) is a useful chiral building block in the synthesis of pharmaceuticals. An NADH-dependent alcohol dehydrogenase (AFPDH) isolated from Candida maris catalyzed the reduction of 5-acetylfuro[2,3-c]pyridine (AFP) to (R)-FPH with 100% enantiomeric excess. The gene encoding AFPDH was cloned and sequenced. The AFPDH gene comprises 762 bp and encodes a polypeptide of 27,230 Da. The deduced amino acid sequence showed a high degree of similarity to those of other members of the short-chain alcohol dehydrogenase superfamily. The AFPDH gene was overexpressed in Escherichia coli under the control of the lac promoter. One L of the cultured broth of an E. coli transformant coexpressing AFPDH and the glucose dehydrogenase (GDH) gene reduced 250 g of AFP to (R)-FPH in an organic solvent two-phase system. Under coupling with NADH regeneration using 2-propanol, 1 L of the cultured broth of an E. coli transformant expressing the AFPDH gene reduced 150 g of AFP to (R)-FPH. The optical purity of the (R)-FPH formed was 100% enantiomeric excess under both reaction conditions.

Purification and characterization of an NADH-dependent alcohol dehydrogenase from Candida maris for the synthesis of optically active 1-(pyridyl)ethanol derivatives.

A novel (R)-specific alcohol dehydrogenase (AFPDH) produced by Candida maris IFO10003 was purified to homogeneity by ammonium sulfate fractionation, DEAE-Toyopearl, and Phenyl-Toyopearl, and characterized. The relative molecular mass of the native enzyme was found to be 59,900 by gel filtration, and that of the subunit was estimated to be 28,900 on SDS-polyacrylamide gel electrophoresis. These results suggest that the enzyme is a homodimer. It required NADH as a cofactor and reduced various kinds of carbonyl compounds, including ketones and aldehydes. AFPDH reduced acetylpyridine derivatives, ß-keto esters, and some ketone compounds with high enantioselectivity. This is the first report of an NADH-dependent, highly enantioselective (R)-specific alcohol dehydrogenase isolated from a yeast. AFPDH is a very useful enzyme for the preparation of various kinds of chiral alcohols.

Cloning, sequencing and expression analysis of a gene encoding alcohol oxidase in Paenibacillus sp. AIU 311.

We have cloned a gene encoding an alcohol oxidase (AOD) specific to aldehyde alcohols from Paenibacillus sp. AIU 311. The AOD gene contains an open reading frame consisting of 618 nucleotides corresponding to 205 amino acid residues. The deduced amino acid sequence exhibits a high similarity to that of manganese superoxide dismutases (SODs). We expressed the cloned gene as an active product in Escherichia coli BL21 cells. The productivity (total units per culture broth volume) of the recombinant AOD expressed in E. coli BL21 is 26,000-fold higher than that of AOD in Paenibacillus sp. AIU 311. The recombinant AOD also exhibits aldehyde alcohol oxidase activity and SOD activity. The recombinant cells described in this study have utility for the production of glyoxal from glycolaldehyde.

Purification and characterization of a new aldehyde oxidase from pseudomonas sp. AIU 362.

An aldehyde oxidase exhibiting high activity on glyoxal was purified to an electrophoretically homogenous state from Pseudomonas sp. AIU 362, which was isolated from a soil sample using a methoxyethanol medium. The enzyme oxidized not only glyoxal but also short-chain aliphatic aldehydes and aromatic aldehydes. Thus, this enzyme was classified into the aldehyde oxidase (ALOD) group. However, it was composed of four identical subunits with a molecular mass of 27 kDa, whereas other microbial ALODs were composed of three hetero subunits, and ALODs from plant and animals were composed of two identical subunits. The NH(2)-terminal sequence also showed no similarity to that of other ALODs. These results indicate that ALOD from Pseudomonas sp. AIU 362 is a new aldehyde oxidase. This ALOD was induced by 2-methoxyethanol, methanol or isopropanol.

Superoxide dismutases exhibit oxidase activity on aldehyde alcohols similar to alcohol oxidase from Paenibacillus sp. AIU 311.

The relations between oxidase activity on aldehyde alcohols and superoxide dismutase (SOD) were investigated, since the amino terminal amino acid sequence of alcohol oxidase (AOD) from Paenibacillus sp. AIU 311, which was specific to aldehyde alcohols, exhibited high similarity to those of SODs containing manganese (Mn(2+)-SOD). Paenibacillus AOD had high SOD activity. The SODs containing manganese, iron, or copper and zinc also exhibited oxidase activities on aldehyde alcohols, and the relative values of oxidase activities on aldehyde alcohols to SOD activity of Mn(2+)-SOD were closer to those of Paenibacillus AOD compared with those of the other SODs. Thus, SODs had AOD activity on aldehyde alcohols as another enzyme activity, and the Paenibacillus AOD and Mn(2+)-SOD were classified into a similar group.

Aldehyde oxidase carrying an unusual subunit structure from Pseudomonas sp. MX-058.

Pseudomonas sp. MX-058 produces aldehyde oxidase catalysing glyoxal to glyoxylic acid. Two aldehyde oxidases (F10 and F13) were purified to homogeneity from Pseudomonas sp. MX-058. F10 and F13 had subunit structures, a heterotetramer and heteropentamer respectively. The N-terminal amino acid sequences of all subunits were highly homologous to amino acid sequences of the putative oxidoreductases of Pseudomonas strains. All of these homologous oxidoreductases have a heterotrimer structure consisting of 85-88 (α), 37-39 (ß) and 18-23 (γ) kDa subunits. However, the α-subunits of F10 and F13 might have decomposed into two [80 (α(1)) and 9 kDa (α(2))] and three [58 (α(1')), 22 (α(1″)) and 9 (α(2)) kDa] subunits, respectively, while the ß- and γ-subunits remained intact. Both F10 and F13 show high activity toward several aliphatic and aromatic aldehydes. The aldehyde oxidases of Pseudomonas sp. MX-058 has unique protein structures, α(1)α(2)ßγ for F10 and α(1')α(1″)α(2)ßγ for F13, a heterotetramer and heteropentamer respectively. The enzymes exhibit significantly low activity toward glyoxylic acid compared with glyoxal, which is an advantageous property for glyoxylic acid production from glyoxal.

Purification and characterization of a novel alcohol oxidase from Paenibacillus sp. AIU 311.

An oxidase catalyzing the conversion of glycolaldehyde to glyoxal was purified to the homogeneous state from Paenibacillus sp. AIU 311, and its properties were revealed. This enzyme was specific to glycolaldehyde and glyceraldehyde, and the reaction rates to other alcohols and aldehydes were less than 6% of that of glycolaldehyde. The Km values for glycolaldehyde and glyceraldehyde were estimated to be 13.2 and 7.5 mM, respectively. The glycolaldehyde oxidation was optimum at pH 6.5 and 50 degrees C. The molecular mass of this enzyme was 49 kDa, and it consisted of two identical subunits of 24 kDa. The NH2-terminal sequence was not homologous to those of alcohol oxidases. This is the first report of an oxidase exhibiting high specificity to a hydroxy group of aldehyde alcohols.

Characterization of alcohol oxidase from Aspergillus ochraceus AIU 031.

An alcohol oxidase (AOD) was found from Aspergillus ochraceus AIU 031, and its characteristics were revealed. This enzyme oxidized short-chain primary alcohols and ethylene glycol, and belonged to the same group as AOD from methylotrophic yeast. However, it differed in the following properties. The K(m) value for ethanol was larger and that for ethylene glycol was smaller than those of AODs derived from methylotrophic yeasts. The ethanol oxidation was optimal at pH 5-7 and 50-55 degrees C. The molecular mass of this enzyme was 262 kDa and consisted of four identical subunits of 68 kDa, which were much smaller than those of methylotrophic yeasts.

Highly active mutants of carbonyl reductase S1 with inverted coenzyme specificity and production of optically active alcohols.

A wild type NADPH-dependent carbonyl reductase from Candida magnoliae (reductase S1) has been found not to utilize NADH as a coenzyme. A mutation to exchange the coenzyme specificity in reductase S1 has been designed by computer-aided methods, including three-dimensional structure modeling and in silico screening of enzyme mutants. Site-directed mutagenesis has been used to introduce systematic substitutions of seven or eight amino acid residues onto the adenosine-binding pocket of the enzyme according to rational computational design. The resulting S1 mutants show NADH-dependency and have lost their ability to utilize NADPH as a coenzyme, but retain those catalytic activities. Kinetic parameter V(max) and K(m) values of those mutants for NADH are 1/3- to 1/10-fold those of the wild type enzyme for NADPH. As a model system for industrial production of optically active alcohols, the S1 mutants can be applied to an asymmetric reduction of ketones, cooperating with a coenzyme-regeneration system that uses an NAD-dependent formate dehydrogenase.

Purification and characterization of a yeast carbonyl reductase for synthesis of optically active (R)-styrene oxide derivatives.

Optically active styrene oxide derivatives are versatile chiral building blocks. Stereoselective reduction of phenacyl halide to chiral 2-halo-1-phenylethanol is the key reaction of the most economical synthetic route. Rhodotorula glutinis var. dairenensis IFO415 was discovered on screening as a potent microorganism reducing a phenacyl halide to the (R)-form of the corresponding alcohol. An NADPH-dependent carbonyl reductase was purified to homogeneity through four steps from this strain. The relative molecular mass of the enzyme was estimated to be 40,000 on gel filtration and 30,000 on SDS-polyacrylamide gel electrophoresis. This enzyme reduced a broad range of carbonyl compounds in addition to phenacyl halides. Some properties of the enzyme and preparation of a chiral styrene oxide using the crude enzyme are reported herein.

Microbial synthesis of (R)- and (S)-3,4-dimethoxyamphetamines through stereoselective transamination.

Two soil isolates, Arthrobactersp. KNK168 and Pseudomonas sp. KNK425, aminated 3,4-dimethoxyphenylacetone in presence of sec-butylamine as an amino donor to yield 3,4-dimethoxyamphetamine (DMA) with different enantioselectivities. The former gave (R)-DMA (>99% e.e.) and the latter the (S)-isomer (>99% e.e.).

Purification and characterization of an alpha-haloketone-resistant formate dehydrogenase from Thiobacillus sp. strain KNK65MA, and cloning of the gene.

Thiobacillus sp. strain KNK65MA, which produced an NAD-dependent formate dehydrogenase (FDH) highly resistant to alpha-haloketones, was newly isolated, i.e., the enzyme showed no loss of activity after a 5-h incubation with alpha-haloketones, such as ethyl 4-chloro-3-oxobutanoate. The enzyme was also resistant to SH reagents. The enzyme, purified to homogeneity, was a dimer composed of identical subunits. The specific activity was 7.6 u/mg, and the apparent Km values for formate and NAD+ were 1.6 and 0.048 mM, respectively. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 44,021; this gene was highly expressed in E. coli cells. The deduced amino acid sequence of this FDH had high identity to other bacterial FDHs.

Purification and characterization of formate dehydrogenase from Ancylobacter aquaticus strain KNK607M, and cloning of the gene.

Ancylobacter aquaticus strain KNK607M, which had high NAD-dependent formate dehydrogenase (FDH) activity, was newly isolated. The enzyme, purified to homogeneity, was a dimer composed of identical subunits with a molecular mass of 44 kDa. The specific activity was 9.5 u/mg, and the enzyme was optimum at pH 6.3 and 50 degrees C, most stable at pH 7.0, and stable at 50 degrees C or lower. The apparent Km values for formate and NAD+ were 2.4 and 0.057 mM, respectively. The enzyme was specific to formate and was inhibited by SH reagents and heavy metal ions. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 43,895; this gene was highly expressed in E. coli cells. The FDH had high identity to other FDHs, i.e., those of Pseudomonas, Mycobacterium, Moraxella, and Paracoccus, which were 91.3%, 90.8%, 84.2%, and 82.3%, respectively.