Identification, Characterization, and Application of Three Enoate Reductases from <i>Pseudomonas putida</i> in In Vitro Enzyme Cascade Reactions

Peters, Christin; Kölzsch, Regina; Kadow, Maria; Skalden, Lilly; Rudroff, Florian; Mihovilovic, Marko D.; Bornscheuer, Uwe T.

doi:10.1002/cctc.201300957

Cited by 32 publications

(32 citation statements)

References 40 publications

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“…The ADH (from Lactobacillus kefir ) depends on Mg 2+ , and the other two enzymes require noncovalently attached flavins as redox‐active components (flavin mononucleotide, FMN, for the ERED from Pseudomonas putida , and flavin adenine dinucleotide, FAD, for the BVMO from Acinetobacter calcoaceticus ) . The starting material cyclohexenol ( 1 a ) was converted to ϵ‐caprolactone ( 1 e ) via the intermediates 2‐cyclohexenone ( 1 b ) and cyclohexanone ( 1 c ) in this particular cascade; the transformation was shown to proceed comparably well on several other examples, which include the conversion of (1 S ,5 S )‐carveol ( 2 a ) to the corresponding lactone 2 e (Scheme ).…”

Section: Introductionmentioning

confidence: 99%

Kinetic Modeling of an Enzymatic Redox Cascade In Vivo Reveals Bottlenecks Caused by Cofactors

et al. 2017

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We describe the development of a kinetic model for the simulation and optimization of an in vivo redox cascade in E. coli in which a combination of an alcohol dehydrogenase, an enoate reductase, and a Baeyer–Villiger monooxygenase is used for the synthesis of lactones. The model was used to estimate the concentrations of active enzyme in the sequential biotransformations to identify bottlenecks together with their reasons and how to overcome them. We estimated adapted Michaelis–Menten parameters from in vitro experiments with isolated enzymes and used these values to simulate the change in the concentrations of intermediates and products during the in vivo cascade reactions. Remarkably, the model indicated that the fastest enzyme was rate‐determining because of the unexpectedly low concentration of the active form, which opens up reversible reaction channels towards byproducts. We also provide substantial experimental evidence that a low intracellular concentration of flavin and nicotinamide cofactors drastically decreased the performance of the in vivo cascade drastically.

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Section: Introductionmentioning

confidence: 99%

Kinetic Modeling of an Enzymatic Redox Cascade In Vivo Reveals Bottlenecks Caused by Cofactors

et al. 2017

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“…To provide access to highly optically pure diastereomers, we then investigated the one‐pot reaction starting with the reduction of the α/β‐unsaturated ketone 1 by using the enoate reductases from Saccharomyces carlsbergensis (OYE) or from Pseudomonas putida (XenB). This afforded optically pure ( S )‐ 2 with >99 % ee 18 (Figure S8). Because OYE showed quantitative conversion, this enzyme was used for further investigations.…”

Section: Methodsmentioning

confidence: 99%

Two Subtle Amino Acid Changes in a Transaminase Substantially Enhance or Invert Enantiopreference in Cascade Syntheses

Skalden¹,

Peters²,

Dickerhoff³

et al. 2015

ChemBioChem

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Amine transaminases (ATAs) are powerful enzymes for the stereospecific production of chiral amines. However, the synthesis of amines incorporating more than one stereocenter is still a challenge. We developed a cascade synthesis to access optically active 3-alkyl-substituted chiral amines by combining two asymmetric synthesis steps catalyzed by an enoate reductase and ATAs. The ATA wild type from Vibrio fluvialis showed only modest enantioselectivity (14 % de) in the amination of (S)-3-methylcyclohexanone, the product of the enoate-reductase-catalyzed reaction step. However, by protein engineering we created two variants with substantially improved diastereoselectivities: variant Leu56Val exhibited a higher R selectivity (66 % de) whereas the Leu56Ile substitution caused a switch in enantiopreference to furnish the S-configured diastereomer (70 % de). Addition of 30 % DMSO further improved the selectivity and facilitated the synthesis of (1R,3S)-1-amino-3-methylcyclohexane with 89 % de at 87 % conversion.

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Section: Formation Of Lactones From Acyclic Precursorsmentioning

confidence: 99%

“…In a subsequent study, three ene-reductases from Pseudomonas putida were used in a very similar cascade, in this case implemented using isolated enzymes (purified enzymes and crude cell-free extracts) [89]. Cyclohexenol, cyclohexanone, and cyclopentenone were investigated, leading to δ-valerolactone or Phenylalanine and derivatives thereof were deracemized by a combination of E. coli whole cells expressing an L-amino acid deaminase and an engineered D-amino acid dehydrogenase to prepare optically pure D-phenylalanines (Scheme 13) [66].…”

Section: Formation Of Lactones From Acyclic Precursorsmentioning

confidence: 99%

Artificial Biocatalytic Linear Cascades to Access Hydroxy Acids, Lactones, and α- and β-Amino Acids

2018

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α-, β-, and ω-Hydroxy acids, amino acids, and lactones represent common building blocks and intermediates for various target molecules. This review summarizes artificial cascades published during the last 10 years leading to these products. Renewables as well as compounds originating from fossil resources have been employed as starting material. The review provides an inspiration for new cascade designs and may be the basis to design variations of these cascades starting either from alternative substrates or extending them to even more sophisticated products.

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Identification, Characterization, and Application of Three Enoate Reductases from Pseudomonas putida in In Vitro Enzyme Cascade Reactions

Cited by 32 publications

References 40 publications

Kinetic Modeling of an Enzymatic Redox Cascade In Vivo Reveals Bottlenecks Caused by Cofactors

Kinetic Modeling of an Enzymatic Redox Cascade In Vivo Reveals Bottlenecks Caused by Cofactors

Two Subtle Amino Acid Changes in a Transaminase Substantially Enhance or Invert Enantiopreference in Cascade Syntheses

Artificial Biocatalytic Linear Cascades to Access Hydroxy Acids, Lactones, and α- and β-Amino Acids

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