Expression, purification, crystallization and X-ray analysis of 3-quinuclidinone reductase from Agrobacterium tumefaciens.

Acta Crystallogr Sect F Struct Biol Cryst Commun

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.

Published: October 2012


Category Ranking

98%

Total Visits

921

Avg Visit Duration

2 minutes

Citations

20

Article Abstract

(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)).

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497986PMC
http://dx.doi.org/10.1107/S1744309112034951DOI Listing

Publication Analysis

Top Keywords

3-quinuclidinone reductase
8
reductase agrobacterium
8
agrobacterium tumefaciens
8
expression purification
4
purification crystallization
4
crystallization x-ray
4
x-ray analysis
4
3-quinuclidinone
4
analysis 3-quinuclidinone
4
tumefaciens r-3-quinuclidinol
4

Similar Publications

Discovery and characterization of the first hyperthermophilic 3-quinuclidinone reductase from hot-spring metagenomes.

Int J Biol Macromol

May 2025

Chongqing Key Laboratory of Functional Substances from Distinctive Medicinal Plants in the Three Gorges Reservoir Area, School of Biological and Chemical Engineering, Chongqing University of Education, Chongqing 400067, China. Electronic address:

Extremozymes play an essential part in the biosynthesis of pharmaceuticals (intermediates). In this study, a mixed assembly was performed for five metagenomes from hot springs. Via a series of procedures, including homology analysis, heterologous expression, and catalytic property characterization, the first hyperthermophilic 3-quinuclidinone reductase (SbQR) was successfully identified.

View Article and Find Full Text PDF

Characterization of a novel 3-quinuclidinone reductase possessing remarkable thermostability.

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 PDF

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

Biotechnol 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 PDF

DiRect: Site-directed mutagenesis method for protein engineering by rational design.

Biochem 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, Kana

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 PDF

Facile fabrication of 3D porous hybrid sphere by co-immobilization of multi-enzyme directly from cell lysates as an efficient and recyclable biocatalyst for asymmetric reduction with coenzyme regeneration in situ.

Int 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 PDF