Photon Factory Activity Report 2005 #23 Part B (2006)
Crystallography

5A,6A,NW12/2003G305

X-ray Structures of Sporobolomyces salmonicolor Aldehyde Reductase 2 andIts Complex with NADPH
Shigehiro KAMITORI*1, Mitsugu YAMADA1, Hiromi YOSHIDA1, Keiko KITA2
1
Information Technology Center and Faculty of Medicine, Kagawa University
1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 791-0793, Japan
2
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto UniversityUji, Kyoto 611-0011, Japan
Introduction
The red yeast Sporobolomyces salmonicolor AKU4429
produces three aldehyde reductases, ARI (323 aminoacids, MW= 35,232 Da), ARII (344 amino acids, MW=37,317 Da) and ARIII (MW= 37,000 Da). ARII canreduce a number of aliphatic and aromatic aldehydes, andketones, such as camphorquinone and benzaldehydes.
The amino acid sequence of ARII is different from that of
the other enzymes belonging to the aldo-keto reductase
superfamily. To obtain new insights into the classificationof ARII and catalytic mechanism underlying thestereoselective reduction of ARII, a three-dimensional
structure needs to be elucidated. The crystal structures ofunliganded ARII (ARII) and the ARII/NADPH complex(ARII/NADPH).

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with 14% of the amino acids. The structure of ARII canbe divided into large domain and small domain, as shownin Figure1. The large domain with the α/β structure is
expected to be a NAD(P)H-binding domain and the small
domain with five α-helices and two small β-sheets
covering the bound NADPH, is expected to be a
substrate-binding domain. With the binding of NADPH,
structural changes occur in the loop region (Ile91-Tyr101)
of the large domain and the loop region (Pro216-Ser222)of the small domain, called as mobile regions, asindicated by red in Figure1. ARII can be classified in theshort chain dehydrogenase reductase (SDR) family basedon three dimensional structure.
ARII has stereoselectivity in its catalytic reaction,
giving rise to excessive production of (S)-alcohols fromethyl 4-chloro-3-oxobutanoate. The X-ray structure ofthe ARII/NADPH complex and preliminary modelingshow that the formation of the hydrophobic channel
induced by the binding of NADPH is closely related tothe stereoselective reduction by ARII [1].

Materials and Methods
A crystal of ARII was grown by the vapor diffusion
method using a protein solution (30 mg/ml ARII in 20
mM Tris-HCl (pH 8.0)) and a reservoir solution (32%(w/v) polyethylene glycol 2000 MME, 100 mM
ammonium sulfate and 200 mM sodium acetate (pH 5.0)).
A crystal of SeMet ARII was obtained under the same
conditions. A crystal of ARII/NADPH was obtained by aco-crystallization method, using the protein solution
containing 4 mM of NADPH and the same reservoirsolution. X-ray diffraction data were collected using anADSC/CCD detector system on the 5A, 6A and NW12beam lines in the Photon Factory. Initial SAD phasing at2.0 Å of SeMet ARII was performed by locating six
selenium sites in the peak dataset using the program
SOLVE. After electron density modification, 60% of
amino acid residues could be located in the resultant
electron density map, using the program RESOLVE.
Further model building was performed with the program
X-fit in the XtalView program system, and the structure
was refined using the program CNS. Using the structureof SeMet ARII, the structure of ARII was determined byisomorphous replacement and refined at a resolution of1.8 Å. The structure of ARII/NADPH was determined bymolecular replacement using the structure of ARII, andrefined at a resolution of 1.6 Å [1].

Figure1. The overall structure of ARII/NADPH complex
illustrated by the programs MOLSCRIPT and Raster3D.
A β-sheet, α-helices on one side of a β-sheet and αhelices on the other side of a β-sheet in the NADPHbinding domain (large domain) are yellow, green andcyan, respectively. The substrate-binding domain (smalldomain) is shown in light magenta. The bound NADPH isshown by a ball-and-stick model.

Results and Discussion
ARII has 12 α-helices with 34% of the amino acidresidues and 12 β-strands forming three β-sheet structures

References[1] S. Kamitori et al., J. Mol. Biol. 353, 551 (2005).
*kamitori@med.kagawa-u.ac.jp

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