Purification and Characterization of a Novel (<i>R</i>)-Imine Reductase from<i>Streptomyces</i>sp. GF3587

Mitsukura, Koichi; Suzuki, Mai; Shinoda, Sho; Kuramoto, Tatsuya; Yoshida, Toyokazu; Nagasawa, Tôru

doi:10.1271/bbb.110303

Cited by 129 publications

(108 citation statements)

References 15 publications

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“…Several enzymes capable of asymmetric reduction of CN bonds have already been identified, including dihydrofolate reductases, thiomorpholine‐carboxylate dehydrogenase, thiazolinyl imine reductase (IRP3) and the Δ 1 ‐pyrroline‐2‐carboxylate reductase . Only recently, a new class of NADPH‐dependent imine reductases (IREDs) has been identified, which show promising activities and high stereoselectivity for the production of a range of synthetically useful amines . Mitsukura et al .…”

Section: Introductionmentioning

confidence: 99%

“…were the first to purify and biochemically characterize R ‐ and S ‐ selective IREDs from Streptomyces sp. GF3587 ( R ‐IRED‐ Ss ) and GF3546 ( S ‐IRED‐ Ss ), respectively . Since then, further members of this family have been biochemically characterized including R ‐IREDs from Streptomyces kanamyceticus ( R ‐IRED‐ Sk ), Streptosporangium roseum ( R ‐IRED‐ Sr ), Streptomyces turgidiscabies ( R ‐IRED‐ St ), and Streptomyces ipomoeae ( R ‐IRED‐ Si ) as well as S ‐IREDs from Paenibacillus elgii ( S ‐IRED‐ Pe ), Streptomyces aurantiacus ( S ‐IRED‐ Sa ), Bacillus cereus ( S ‐IRED‐ Bc ), and Nocardiopsis halophila ( S ‐IRED‐ Nh ) .…”

Section: Introductionmentioning

confidence: 99%

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Identification of imine reductase‐specific sequence motifs

et al. 2016

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Chiral amines are valuable building blocks for the production of a variety of pharmaceuticals, agrochemicals and other specialty chemicals. Only recently, imine reductases (IREDs) were discovered which catalyze the stereoselective reduction of imines to chiral amines. Although several IREDs were biochemically characterized in the last few years, knowledge of the reaction mechanism and the molecular basis of substrate specificity and stereoselectivity is limited. To gain further insights into the sequence-function relationships, the Imine Reductase Engineering Database (www.IRED.BioCatNet.de) was established and a systematic analysis of 530 putative IREDs was performed. A standard numbering scheme based on R-IRED-Sk was introduced to facilitate the identification and communication of structurally equivalent positions in different proteins. A conservation analysis revealed a highly conserved cofactor binding region and a predominantly hydrophobic substrate binding cleft. Two IRED-specific motifs were identified, the cofactor binding motif GLGxMGx(5 )[ATS]x(4) Gx(4) [VIL]WNR[TS]x(2) [KR] and the active site motif Gx[DE]x[GDA]x[APS]x(3){K}x[ASL]x[LMVIAG]. Our results indicate a preference toward NADPH for all IREDs and explain why, despite their sequence similarity to β-hydroxyacid dehydrogenases (β-HADs), no conversion of β-hydroxyacids has been observed. Superfamily-specific conservations were investigated to explore the molecular basis of their stereopreference. Based on our analysis and previous experimental results on IRED mutants, an exclusive role of standard position 187 for stereoselectivity is excluded. Alternatively, two standard positions 139 and 194 were identified which are superfamily-specifically conserved and differ in R- and S-selective enzymes.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of imine reductase‐specific sequence motifs

et al. 2016

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“…They discovered two strains, Streptomyces GF3587 and Streptomyces GF3546, which catalysed the reduction of 1 to the 2‐methylpyrrolidine (amine) products ( R )‐ 2 and ( S )‐ 2 , respectively, with high enantiomeric excess. The Mitsukura group purified the enzymes responsible for these stereocomplementary reductions—“3587‐IRED” (Uniprot code M4ZRJ3) and “3546‐IRED” (M4ZS15), respectively—and in subsequent papers also reported the cloning and heterologous expression in Escherichia coli of the genes that encode them 12. 14 These IREDs were shown to be dependent for activity exclusively on the nicotinamide cofactor NADPH, and to exist in solution as homodimers of monomers, each of approximately 30 kDa.…”

Section: Introductionmentioning

confidence: 99%

Structure, Activity and Stereoselectivity of NADPH‐Dependent Oxidoreductases Catalysing the S‐Selective Reduction of the Imine Substrate 2‐Methylpyrroline

et al. 2015

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Oxidoreductases from Streptomyces sp. GF3546 [3546-IRED], Bacillus cereus BAG3X2 (BcIRED) and Nocardiopsis halophila (NhIRED) each reduce prochiral 2-methylpyrroline (2MPN) to (S)-2-methylpyrrolidine with >95 % ee and also a number of other imine substrates with good selectivity. Structures of BcIRED and NhIRED have helped to identify conserved active site residues within this subgroup of imine reductases that have S selectivity towards 2MPN, including a tyrosine residue that has a possible role in catalysis and superimposes with an aspartate in related enzymes that display R selectivity towards the same substrate. Mutation of this tyrosine residue-Tyr169-in 3546-IRED to Phe resulted in a mutant of negligible activity. The data together provide structural evidence for the location and significance of the Tyr residue in this group of imine reductases, and permit a comparison of the active sites of enzymes that reduce 2MPN with either R or S selectivity.

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“…One challenge is that both reduction and oxidation have to take place under the same reaction conditions, but the pH-optima and reaction rates for both reactions differ significantly. For many of the IREDs it could be shown that the maximum specific activity for oxidizing a cyclic amine is 2-30 times smaller compared to the reduction of its corresponding imine (Gand et al, 2014;Mitsukura et al, 2011) (each measured at the respective pH optimum). As a proof-of-principle for the one-enzyme approach, we investigated the reduction of 2-methylpyrroline 1 as a well-known model substrate.…”

Section: Cofactor Recycling By Coupled Oxidative Deaminationmentioning

confidence: 99%

“…These enzymes allow two "types" of reactions: (i) the reduction of a cyclic imine to a cyclic sec-amine or (ii) the reductive amination of a ketone with a primary amine yielding a sec-amine, where a proof of concept (Huber et al, 2014;Scheller et al, 2015) and preparative applications were demonstrated very recently (Wetzl et al, 2016). In the previous years, several research groups discovered a large number of IREDs (Gand et al, 2014;Huber et al, 2014;Li et al, 2015;Man et al, 2015;Mitsukura et al, 2013Mitsukura et al, , 2011Mitsukura et al, , 2010Rodríguez-Mata et al, 2013;Scheller et al, 2015;Wetzl et al, 2015). In addition, an imine reductase engineering database with more than 1000 putative IREDs is established (Scheller et al, 2014).…”

Section: Introductionmentioning

confidence: 99%