Enzymatic synthesis of N-methyl-l-phenylalanine by a novel enzyme, N-methyl-l-amino acid dehydrogenase, from Pseudomonas putida

Muramatsu, Hisashi; Mihara, Hisaaki; Kakutani, Ryo; Yasuda, Masato; Ueda, Makoto; Kurihara, Tatsuo; Esaki, Nobuyoshi

doi:10.1016/j.tetasy.2004.06.030

Cited by 29 publications

(21 citation statements)

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“…The dpkA Gene Is Essential for the Catabolism of D-Lysine and D-Proline-We found that a novel enzyme, N-methyl-Lamino acid dehydrogenase, is encoded by a gene named dpkA in P. putida ATCC12633 (19). However, this bacterium was unable to grow on N-methyl-L-alanine as a sole carbon source (Table I), and we speculated that the gene has other physiological functions than the metabolism of N-methyl-L-amino acids.…”

Section: Resultsmentioning

confidence: 99%

“…All other reagents were of analytical grade from Nacalai Tesque (Kyoto, Japan) and Wako Pure Chemical Industries (Osaka, Japan). DpkA was purified from Escherichia coli BL21(DE3) carrying pDPKA as described previously (19).…”

Section: Methodsmentioning

confidence: 99%

“…We searched for a new enzyme catalyzing the formation of N-methyl-L-phenylalanine from phenylpyruvate and methylamine and found that the enzyme gene is identical to the gene annotated as MDH of the new family of NAD(P)-dependent dehydrogenases described above (19). However, the gene product showed no MDH activity.…”

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The Putative Malate/Lactate Dehydrogenase from Pseudomonas putida Is an NADPH-dependent Δ1-Piperideine-2-carboxylate/Δ1-Pyrroline-2-carboxylate Reductase Involved in the Catabolism of d-Lysine and d-Proline

Muramatsu

Mihara

Kakutani

et al. 2005

Journal of Biological Chemistry

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A Pseudomonas putida ATCC12633 gene, dpkA, encoding a putative protein annotated as malate/L-lactate dehydrogenase in various sequence data bases was disrupted by homologous recombination. The resultant dpkA ؊ mutant was deprived of the ability to use D-lysine and also D-proline as a sole carbon source. The dpkA gene was cloned and overexpressed in Escherichia coli, and the gene product was characterized. The enzyme showed neither malate dehydrogenase nor lactate dehydrogenase activity but catalyzed the NADPH-dependent reduction of such cyclic imines as ⌬ 1 -piperideine-2-carboxylate and ⌬ 1 -pyrroline-2-carboxylate to form L-pipecolate and L-proline, respectively. NADH also served as a hydrogen donor for both substrates, although the reaction rates were less than 1% of those with NADPH. The reverse reactions were also catalyzed by the enzyme but at much lower rates. Thus, the enzyme has dual metabolic functions, and we named the enzyme ⌬ 1 -piperideine-2-carboxylate/⌬ 1 -pyrroline-2-carboxylate reductase, the first member of a novel subclass in a large family of NAD(P)-dependent oxidoreductases.Lactate dehydrogenase (LDH) 1 and malate dehydrogenase (MDH) comprise a complex protein superfamily with multiple enzyme homologs found in eubacteria, Archaea, and eukaryotes (1). They catalyze NAD(P)-dependent interconversions between lactate and pyruvate and between malate and oxaloacetate, respectively. However, a new class of NAD(P)-dependent malate/L-lactate dehydrogenases with no sequence homology to "orthodox" MDH or LDH has been demonstrated (2, 3). Moreover, they have no Rossmann fold, which is a sixstranded parallel ␤-sheet core surrounded on both sides by helices (4), in their NAD(P)-binding domains in contrast to the orthodox proteins. Consequently, the new class of malate/Llactate dehydrogenase family has been distinguished from the orthodox one as shown in protein data bases such as InterPro (www.ebi.ac.uk/interpro/index.html) (5) and Pfam (www.sanger. ac.uk/Software/Pfam/) (6). However, many of the family members are annotated without functional evidence as MDH or LDH only because of their sequence similarities to those of a few enzymes such as MDH from Methanothermus fervidus (2) and LDH from Alcaligenes eutrophus (3). In fact, three hypothetical MDHs of this family have been shown to be (S)-2-hydroxyacid dehydrogenase (7), ureidoglycolate dehydrogenase (8), and 2,3-diketo-L-gulonate reductase (9). The NAD(P) dependence is common to them, but no other functional similarities can be assigned among them. Thus, we expect the occurrence of various other proteins with new functions in this family even though they are annotated as MDH (or LDH) in protein data bases.Pseudomonas strains use both enantiomers of lysine as a sole source of carbon (as well as nitrogen) (10, 11). L-Lysine is catabolized by Pseudomonas putida through the ␦-aminovalerate pathway (11), whereas D-lysine is catabolized through the pipecolate pathway involving a series of reactions through six-carbon cyclic intermediates (12, 13) (Fig. 1)...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Putative Malate/Lactate Dehydrogenase from Pseudomonas putida Is an NADPH-dependent Δ1-Piperideine-2-carboxylate/Δ1-Pyrroline-2-carboxylate Reductase Involved in the Catabolism of d-Lysine and d-Proline

Muramatsu

Mihara

Kakutani

et al. 2005

Journal of Biological Chemistry

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“…The results for the -amino acids 165a-i utilized in this study are presented in Table [57,58]. [61]. Using this methodology it is possible to reduce the amount of triethylsilane to 2 equiv., as compared to 3 equiv.…”

Section: Novel Proline Derivativesmentioning

confidence: 99%

Recent Advances in the Synthesis of Unnatural α-Amino Acids

Perdih¹,

Dolenc

2007

COC

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The synthesis of unnatural -amino acids is an area of research that has gained a lot of attention in recent years. The availability of synthetic methods for novel unnatural amino acid derivatives and related compounds will be a critical point in the de novo design of molecules that mimic the conformation of the natural, active peptides. These molecules (peptidomimetics) are designed to display high receptor affinity and selectivity, in addition to enhanced bioavailability and metabolic stability.This review focuses on selected recent synthetic methodologies leading to unnatural -amino acids including chiral catalysts that enable enantioselective synthesis and microwave-assisted synthesis. Solid phase synthesis and construction of organometallic amino acids reveal the scope of influence that this field has in organic chemistry. Additionally, the article is aimed to provide a brief insight into the biosynthetic approaches to the synthesis of -amino acid derivatives, and to give the reader an appreciation of the field.

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“…This suggests that cyclization occurs in the active site of the enzyme before release of the product into solution, which is consistent with an observation by Danson et al 24) Previously we found that Pip2C reductase from P. putida also has N-methyl-Lamino acid dehydrogenase activity 21) and can be used for the synthesis of N-methyl-L-phenylalanine. 25) Thus the enzyme is unique in that it is useful for the production of different types of chiral compounds. Production of L-pipecolic acid was performed at 30 C at pH 7.5 in a reaction mixture containing L-lysine (55 mM), glucose (550 mM), NADP þ (0.11 mM), FAD (1 mM), L-lysine -oxidase (3.0 U/ml), catalase (50 U/ml), Tris-HCl (100 mM), and a cell-free extract (3.0 mg-protein/ml).…”

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Enzymatic Synthesis ofL-Pipecolic Acid by Δ¹-Piperideine-2-carboxylate Reductase fromPseudomonas putida

Muramatsu

Mihara

Yasuda

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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L-Pipecolic acid is a chiral pharmaceutical intermediate. An enzymatic system for the synthesis of L-pipecolic acid from L-lysine by commercial L-lysine -oxidase from Trichoderma viride and an extract of recombinant Escherichia coli cells coexpressing Á 1 -piperideine-2-carboxylate reductase from Pseudomonas putida and glucose dehydrogenase from Bacillus subtilis is described. A laboratory-scale process provided 27 g/l of Lpipecolic acid in 99.7% e.e.Key words: Á 1 -piperideine-2-carboxylate reductase; Pseudomonas putida; L-pipecolic acid; Á 1 -piperideine-2-carboxylate L-Pipecolic acid, a nonproteinogenic -amino acid, is a key component of many bioactive molecules, such as the immunosuppressant FK506, 1) the anticancer agent VX710, 2) the antifungal antibiotic demethoxyrapamycin,3) the N-methyl-D-aspartate antagonist selfotel, 4) the antitumor antibiotic sandramycin, 5) the phytotoxic metabolite Cyl-2, 6) the anesthetic bupivacaine, 7) and the HIV protease inhibitor palinavir.8) There is an increasing demand for a convenient and efficient synthetic route to enantiomerically pure L-pipecolic acid because of its use in the synthesis of new medicaments.Current methods to obtain a pure enantiomer of L-pipecolic acid involve chemical resolution, 9) stereoselective transformation, 10) the derivatization of natural amino acids, 11) and enzymatic reactions, [12][13][14][15][16] but most of these methods fail to provide a satisfactory solution for the synthesis of chiral pipecolic acid on an industrial scale due to certain limitations, such as tedious procedures, low yields, and unavailability of starting materials. Hence, new and convenient methods for the preparation of optically active pipecolic acid are required.Recently, we identified and cloned the gene encoding17) The enzyme, which belongs to a new NAD(P)H-dependent oxidoreductase family, 18,19) catalyzes the reduction of Pip2C to L-pipecolic acid and is involved in D-lysine catabolism. 17) In this paper, we describe the production of L-pipecolic acid by an enzyme-coupled system consisting of Pip2C reductase, glucose dehydrogenase (GDH) from Bacillus subtilis, 20) and commercial L-lysine -oxidase from Trichoderma viride (Yamasa, Hiroshima, Japan) (Scheme 1).Recombinant Escherichia coli BL21(DE3) cells harboring both pDPKA, 17,21) which carries a gene for Pip2C reductase, and pSTVbsGDH, which has a GDH 20) gene between the EcoRI and PstI sites of pSTV28 (Takara Shuzo, Japan), were cultured in Luria-Bertani medium containing 100 mg/ml ampicillin and 25 mg/ml chloramphenicol at 37 C for 16 h. Three h after induction of gene expression with 1 mM isopropyl--D-thiogalactopyranoside, the cells were harvested and washed twice with 20 mM Tris-HCl (pH 7.0). The washed cells were disrupted by sonication and centrifuged. The resulting crude extract was used directly for the production of L-pipecolic acid. The standard reaction mixture for the production of L-pipecolic acid contained L-lysine (55 mM), glucose (550 mM), NADPL-lysine -oxidase (Yamasa, 3.0 U/ml), catalase from b...

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Enzymatic synthesis of N-methyl-l-phenylalanine by a novel enzyme, N-methyl-l-amino acid dehydrogenase, from Pseudomonas putida

Cited by 29 publications

References 14 publications

The Putative Malate/Lactate Dehydrogenase from Pseudomonas putida Is an NADPH-dependent Δ1-Piperideine-2-carboxylate/Δ1-Pyrroline-2-carboxylate Reductase Involved in the Catabolism of d-Lysine and d-Proline

The Putative Malate/Lactate Dehydrogenase from Pseudomonas putida Is an NADPH-dependent Δ1-Piperideine-2-carboxylate/Δ1-Pyrroline-2-carboxylate Reductase Involved in the Catabolism of d-Lysine and d-Proline

Recent Advances in the Synthesis of Unnatural α-Amino Acids

Enzymatic Synthesis ofL-Pipecolic Acid by Δ¹-Piperideine-2-carboxylate Reductase fromPseudomonas putida

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Enzymatic synthesis of N-methyl-l-phenylalanine by a novel enzyme, N-methyl-l-amino acid dehydrogenase, from Pseudomonas putida

Cited by 29 publications

References 14 publications

The Putative Malate/Lactate Dehydrogenase from Pseudomonas putida Is an NADPH-dependent Δ1-Piperideine-2-carboxylate/Δ1-Pyrroline-2-carboxylate Reductase Involved in the Catabolism of d-Lysine and d-Proline

The Putative Malate/Lactate Dehydrogenase from Pseudomonas putida Is an NADPH-dependent Δ1-Piperideine-2-carboxylate/Δ1-Pyrroline-2-carboxylate Reductase Involved in the Catabolism of d-Lysine and d-Proline

Recent Advances in the Synthesis of Unnatural α-Amino Acids

Enzymatic Synthesis ofL-Pipecolic Acid by Δ1-Piperideine-2-carboxylate Reductase fromPseudomonas putida

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Enzymatic Synthesis ofL-Pipecolic Acid by Δ¹-Piperideine-2-carboxylate Reductase fromPseudomonas putida