Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko; Takeshita, Daijiro; Kumashiro, Shoko; Uzura, Atsuko; Urano, Nobuyuki; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

doi:10.1016/j.bbrc.2014.03.030

Cited by 12 publications

(9 citation statements)

References 22 publications

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“…The chiral alcohol (R)-3-quinuclidinol was selected as the target compound for these studies, as it is a key component in a number of drug molecules, such as talsaclidine, cevimeline and solifenacin 38,39 . In order to synthesise the chiral alcohol, a quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was expressed in Escherichia coli and purified accordingly (Supplementary Methods) 40,41 . When supplied with NADH and 3-quinuclidinone, the AtQR is capable of producing (R)-3-quinuclidinol with >99% ee.…”

Section: H 2 -Driven Biocatalytic System For Generation Of [4-2 H]-nadhmentioning

confidence: 99%

Bringing biocatalytic deuteration into the toolbox of asymmetric isotopic labelling techniques

et al. 2020

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Enzymes dependent on nicotinamide cofactors are important components of the expanding range of asymmetric synthetic techniques. New challenges in asymmetric catalysis are arising in the field of deuterium labelling, where compounds bearing deuterium ( 2 H) atoms at chiral centres are becoming increasingly desirable targets for pharmaceutical and analytical chemists. However, utilisation of NADH-dependent enzymes for 2 H-labelling is not straightforward, owing to difficulties in supplying a suitably isotopically-labelled cofactor ([4-2 H]-NADH). Here we report on a strategy that combines a clean reductant (H 2 ) with a cheap source of 2 H-atoms ( 2 H 2 O) to generate and recycle [4-2 H]-NADH. By coupling [4-2 H]-NADH-recycling to an array of C=O, C=N, and C=C bond reductases, we demonstrate asymmetric deuteration across a range of organic molecules under ambient conditions with near-perfect chemo-, stereo-and isotopic selectivity. We demonstrate the synthetic utility of the system by applying it in the isolation of the heavy drug (1S,3'R)-[2',2',3'-2 H 3 ]solifenacin fumarate on a preparative scale.

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Section: H 2 -Driven Biocatalytic System For Generation Of [4-2 H]-nadhmentioning

confidence: 99%

Bringing biocatalytic deuteration into the toolbox of asymmetric isotopic labelling techniques

et al. 2020

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“…2(C)), its tertiary amine is exposed to solvent, while on the opposite side, hydrophobic hydrocarbons are adjacent to the hydrophobic wall composed of Ile167 and Phe212. 19) The alanine mutation of these hydrophobic residues completely lost the catalytic activity, indicating that the residues are essential for the substrate binding at the active site. The K m value of RrQR is very high toward 3-quinuclidinone (440 mM).…”

Section: Structural Basis For the Stereoselectivity Of Quinuclidinmentioning

confidence: 99%

“…2(C) and (D)). 19,20) In the structure of RrQR, Ser166 forms a hydrogen bond with the carbonyl oxygen of 3-quinuclidinone. Hydride is transferred to the carbonyl carbon of the substrate from the nicotinamide ring of NADPH.…”

Section: Structural Basis For the Stereoselectivity Of Quinuclidinmentioning

confidence: 99%

“…X-ray crystallographic studies of these enzymes have revealed the unique mechanisms underlying their strict stereoselectivity and enhanced substrate-binding affinity upon cofactor binding. 19,20) Both quinuclidinone reductases share a core (or large) domain composed of a typical Rossmann fold with a seven-stranded parallel β-sheet and seven α-helices, and form a tightly associated homotetramer. 19,20) Although the cofactor-binding site is located at the core domain in both reductases, RrQR and AtQR exhibit different cofactor selectivities toward NADPH and NADH, respectively.…”

Section: Structural Basis For the Stereoselectivity Of Quinuclidinmentioning

confidence: 99%

“…19,20) Both quinuclidinone reductases share a core (or large) domain composed of a typical Rossmann fold with a seven-stranded parallel β-sheet and seven α-helices, and form a tightly associated homotetramer. 19,20) Although the cofactor-binding site is located at the core domain in both reductases, RrQR and AtQR exhibit different cofactor selectivities toward NADPH and NADH, respectively. The selectivity is defined by the residues surrounding the ribose ring of the adenosine moiety ( Fig.…”

Section: Structural Basis For the Stereoselectivity Of Quinuclidinmentioning

confidence: 99%

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Structural analysis of enzymes used for bioindustry and bioremediation

Tanokura

Miyakawa

Guan

et al. 2015

Bioscience, Biotechnology, and Biochemistry

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Microbial enzymes have been widely applied in the large-scale, bioindustrial manufacture of food products and pharmaceuticals due to their high substrate specificity and stereoselectivity, and their effectiveness under mild conditions with low environmental burden. At the same time, bioremedial techniques using microbial enzymes have been developed to solve the problem of industrial waste, particularly with respect to persistent chemicals and toxic substances. And finally, structural studies of these enzymes have revealed the mechanistic basis of enzymatic reactions, including the stereoselectivity and binding specificity of substrates and cofactors. The obtained structural insights are useful not only to deepen our understanding of enzymes with potential bioindustrial and/or bioremedial application, but also for the functional improvement of enzymes through rational protein engineering. This review shows the structural bases for various types of enzymatic reactions, including the substrate specificity accompanying cofactor-controlled and kinetic mechanisms.

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Converting the 3‐quinuclidinone reductase from Agrobacterium tumefaciens into the ethyl 4‐chloroacetoacetate reductase by site‐directed mutagenesis

Liu

Gou

Bai

et al. 2021

Biotech and App Biochem

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In this study, the 3‐quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site‐directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single‐point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4‐chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the seven variants could asymmetrically reduce COBE to ethyl (S)‐4‐chloro‐3‐hydroxybutyrate ((S)‐CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40°C, and the catalytic efficiency (kcat/Km) for COBE was 51.36 s–1·mM–1. This study showed that the substrate specificity of AtQR could be changed through site‐directed mutagenesis at the residue 197.

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Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

Cited by 12 publications

References 22 publications

Bringing biocatalytic deuteration into the toolbox of asymmetric isotopic labelling techniques

Bringing biocatalytic deuteration into the toolbox of asymmetric isotopic labelling techniques

Structural analysis of enzymes used for bioindustry and bioremediation

Converting the 3‐quinuclidinone reductase from Agrobacterium tumefaciens into the ethyl 4‐chloroacetoacetate reductase by site‐directed mutagenesis

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