(R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72 Å resolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P2(1), with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 Å, β = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V(M) = 2.08 Å(3) Da(-1)).
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http://dx.doi.org/10.1107/S1744309112034951 | DOI Listing |
Int J Biol Macromol
April 2024
Chongqing Key Laboratory of Medicinal Resources in the Three Gorges Reservoir Region, School of Biological and Chemical Engineering, Chongqing University of Education, Chongqing 400067, China. Electronic address:
The 3-quinuclidinone reductase plays an irreplaceable role in the biopreparation of (R)-3-quinuclidinol, an intermediate vital for synthesis of various pharmaceuticals. Thermal robustness is a critical factor for enzymatic synthesis in industrial applications. This study characterized a new 3-quinuclidinone reductase, named SaQR, with significant thermal stability.
View Article and Find Full Text PDFBiotechnol Appl Biochem
August 2022
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China.
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.
View Article and Find Full Text PDFBiochem Biophys Res Commun
April 2021
Laboratory for Cellular Structural Biology, RIKEN Center for Biosystems Dynamics Research (BDR), 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan; Laboratory for Biomolecular Structure and Dynamics, RIKEN Quantitative Biology Center (QBiC), 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan. Electronic address:
Site-directed mutagenesis (SDM), an indispensable method in molecular biology and protein engineering, is rather time-consuming and laborious. Protein engineering, especially that of enzymes, nowadays increasingly relies on rational design approaches in which both SDM and protein expression are the bottlenecks because they are generally based on the recombinant DNA technology. Here, we developed a new PCR-based mutagenesis method, DiRect, that achieves high performance in product quality (≥99% substitution) without recombinant DNA technology.
View Article and Find Full Text PDFInt J Biol Macromol
October 2017
Department of Medicinal Chemistry, School of Pharmacy, Chongqing Medical University, Chongqing 400016, PR China; Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, Chongqing Medical University, Chongqing 400016, PR China. Electronic address:
Ni-agarose bead-wrapped multi-enzyme/inorganic hybrid sphere composed of the immobilized enzymes as organic component and NaHPO and NaCl as inorganic component was developed by co-immobilizing extracellular His-tagged 3-quinuclidinone reductases and glucose dehydrogenase without pre-purification. The resulting biocatalysts has 3D porous architectures as confirmed by SEM and FESEM, and it enabled the continuous biotransformation of 3-quinuclidone to (R)-3-quinuclidinol with cofactor regeneration in situ. The 3D porous biocatalysts were formed via three steps: First, immobilization of the His-tagged enzymes directly from the cell lysates supernatant.
View Article and Find Full Text PDFPhytochemistry
July 2016
Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing 400715, China. Electronic address:
Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally characterised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to tropinone reductase I from TAs-producing herbal plant species.
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