RESUMO
A NADPH-dependent (S)-imine reductase (SIR) was purified to be homogeneous from the cell-free extract of Streptomyces sp. GF3546. SIR appeared to be a homodimer protein with subunits of 30.5 kDa based on SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It also catalyzed the (S)-enantioselective reduction of not only 2-methyl-1-pyrroline (2-MPN) but also 1-methyl-3,4-dihydroisoquinoline and 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline. Specific activities for their imines were 130, 44, and 2.6 nmol min(-1) mg(-1), and their optical purities were 92.7 % ee, 96.4 % ee, and >99 % ee, respectively. Using a NADPH-regenerating system, 10 mM 2-MPN was converted to amine with 100 % conversion and 92 % ee after 24 h. The amino acid sequence analysis revealed that SIR showed about 60 % identity to 6-phosphogluconate dehydrogenase. However, it showed only 37 % identity with Streptomyces sp. GF3587 (R)-imine reductase. Expression of SIR in Escherichia coli was achieved, and specific activity of the cell-free extract was about two times higher than that of the cell-free extract of Streptomyces sp. GF3546.
Assuntos
Aminas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Iminas/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Streptomyces/enzimologia , Aminas/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Expressão Gênica , Iminas/química , Dados de Sequência Molecular , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Homologia de Sequência de Aminoácidos , Microbiologia do Solo , Streptomyces/química , Streptomyces/genética , Streptomyces/isolamento & purificação , Especificidade por SubstratoRESUMO
We discovered a D-phenylserine deaminase that catalyzed the pyridoxal 5'-phosphate (PLP)-dependent deamination reaction from D-threo-phenylserine to phenylpyruvate in newly isolated Arthrobacter sp. TKS1. The enzyme was partially purified, and its N-terminal amino acid sequence was analyzed. Based on the sequence information, the gene encoding the enzyme was identified and expressed in Escherichia coli. The expressed protein was purified to homogeneity and characterized. The enzyme consisted of two identical 46-kDa subunits and showed maximum activity at pH 8.5 and 55°C. The enzyme was stable in the range of pH 7.5 to pH 8.5 and up to 50°C. The enzyme acted on the D-forms of ß-hydroxy-α-amino acids, such as D-threo-phenylserine (K(m), 19 mM), D-serine (K(m), 5.8 mM), and D-threonine (K(m), 102 mM). As L-threonine, D-allo-threonine, L-allo-threonine, and DL-erythro-phenylserine were inert, the enzyme could distinguish D-threo-form from among the four stereoisomers of phenylserine or threonine. The enzyme was activated by ZnSO(4), CuSO(4), BaCl(2), and CoCl(2) and strongly inhibited by phenylhydrazine, sodium borohydride, hydroxylamine, and DL-penicillamine. The enzyme exhibited absorption maxima at 280 and around 415 nm. The enzyme has an N-terminal domain similar to that of alanine racemase, which belongs to the fold type III group of pyridoxal enzymes.
Assuntos
Arthrobacter/enzimologia , Proteínas de Bactérias/química , Liases/química , Serina/análogos & derivados , Sequência de Aminoácidos , Arthrobacter/química , Arthrobacter/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estabilidade Enzimática , Cinética , Liases/genética , Liases/metabolismo , Dados de Sequência Molecular , Peso Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Serina/metabolismo , Especificidade por SubstratoRESUMO
To compare NADH-regeneration systems for the synthesis of (S)-4-chloro-3-hydroxybutanoate (ECHB), a novel NADH-dependent carbonyl reductase (KaCR1), which reduced ethyl 4-chloroacetoacetate (ECAA) to form (S)-ECHB, was screened and purified from Kluyveromyces aestuarii and a gene encoding KaCR1 was cloned. Glucose dehydrogenase (GDH) and formate dehydrogenase (FDH) were compared as enzymes for NADH regeneration using Escherichia coli cells coexpressing each enzyme with KaCR1. E. coli cells coexpressing GDH produced 45.6 g/l of (S)-ECHB from 50 g/l of ECAA and E. coli cells coexpressing FDH, alternatively, produced only 19.0 g/l. The low productivity in the case of FDH was suggested to result from the low activity and instability of FDH.
Assuntos
Acetoacetatos/metabolismo , Oxirredutases do Álcool/metabolismo , Butiratos/metabolismo , Kluyveromyces/enzimologia , NAD/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sequência Conservada/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Formiato Desidrogenases/metabolismo , Glucose 1-Desidrogenase/metabolismo , Dados de Sequência Molecular , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Plasmídeos/genética , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
A novel carbonyl reductase (KLCR1) that reduced ethyl 4-chloroacetoacetate (ECAA) to synthesize ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-ECHB) was purified from Kluyveromyces lactis. KLCR1 catalyzed the NADPH-dependent reduction of ECAA enantioselectively but not the oxidation of (S)-ECHB. From partial amino acid sequences, KLCR1 was suggested to be an alpha subunit of fatty acid synthase (FAS) but did not have FAS activity.
