title: Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR.
abstract: (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 1.72 Å. 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.
authors.affiliation: 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.
authors.forename: Feng
authors.initials: F
authors.lastname: Hou
authors.affiliation: 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.
authors.forename: Takuya
authors.initials: T
authors.lastname: Miyakawa
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531, Japan; Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kataoka
authors.affiliation: 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.
authors.forename: Daijiro
authors.initials: D
authors.lastname: Takeshita
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Kumashiro
authors.affiliation: Research & Development Center, Nagase & Co., Ltd., 2-2-3 Muratani, Nishi-ku, Kobe 651-2241, Japan.
authors.forename: Atsuko
authors.initials: A
authors.lastname: Uzura
authors.affiliation: Division of Applied Life Sciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 559-8531, Japan; Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Urano
authors.affiliation: 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.
authors.forename: Koji
authors.initials: K
authors.lastname: Nagata
authors.affiliation: Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan; Faculty of Bioenvironmental Science, Kyoto Gakuen University, Sogabe-cho, Kameoka 621-8555, Japan.
authors.forename: Sakayu
authors.initials: S
authors.lastname: Shimizu
authors.affiliation: 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. Electronic address: amtanok@mail.ecc.u-tokyo.ac.jp.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
mesh_terms: D016960:Agrobacterium tumefaciens / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011812:Quinuclidines / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D011812:Quinuclidines; D009243:NAD; C090998:3-quinuclidinone; D010088:Oxidoreductases
keywords: Agrobacterium tumefaciens; Crystal structure; Quinuclidinone; Short-chain dehydrogenases/reductases