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

• Published in: Biochemical and biophysical research communications Vol. 446; no. 4; pp. 911 - 915
• Main Authors: Hou, Feng, Miyakawa, Takuya, Kataoka, Michihiko, Takeshita, Daijiro, Kumashiro, Shoko, Uzura, Atsuko, Urano, Nobuyuki, Nagata, Koji, Shimizu, Sakayu, Tanokura, Masaru
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.04.2014
• Subjects: 60 APPLIED LIFE SCIENCES; AFFINITY; Agrobacterium tumefaciens; Agrobacterium tumefaciens - chemistry; Agrobacterium tumefaciens - enzymology; Agrobacterium tumefaciens - metabolism; AMINES; Binding Sites; CRYSTAL STRUCTURE; Crystallography, X-Ray; DRUGS; Models, Molecular; NAD - metabolism; NADH2; OXIDOREDUCTASES; Oxidoreductases - chemistry; Oxidoreductases - metabolism; Protein Conformation; PROTONS; Quinuclidines - metabolism; Quinuclidinone; REDUCTION; RESIDUES; Short-chain dehydrogenases/reductases; STABILITY; Stereoisomerism; Substrate Specificity; SUBSTRATES; SYNTHESIS; Agrobacterium tumefaciens; Short-chain dehydrogenases/reductases; Quinuclidinone; Crystal structure
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2014.03.030

•Crystal structure of AtQR has been determined at 1.72Å.•NADH binding induces the formation of substrate binding site.•AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate.•Additional Glu197 residue is critical to the high binding affinity. (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.