Photon Factory Activity Report 2016 #34 (2017)

AR-NE3A/2016G502, 2017G010

Crystal structure of thermostable NAD(P)H-dependent carbonyl reductase from thehyperthermophilic archaeon Aeropyrum pernix K1
Kazunari Yoneda*1, Haruhiko Sakuraba2, Yudai Fukuda1, Toshihisa Ohshima3
1
Department of Bioscience, School of Agriculture, Tokai University, Kumamoto, 862-8652, Japan2
Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393
Ikenobe, Miki-cho, Kita-gun, Kagawa761-0795, Japan3
Department of Biomedical Engineering, Osaka Institute of Technology, Osaka, Japan
1 Introduction
A gene encoding NAD(P)H-dependent carbonyl
reductase (CR) from the hyperthermophilic archaeon
Aeropyrum pernix K1 was overexpressed in Escherichia
coli. The expressed enzyme was the most thermostable
CR found to date. A. pernix CR catalyzed the reductionof various carbonyl compounds including ethyl 4-chloro3-oxobutanoate and 9,10-phenanthrenequinone, similar tothe thyroidectomised (Tx) CR from chicken fatty liver [1,2]. However, A. pernix CR exhibited significantly higher
Km values against several substrates than Tx chicken fattyliver CR. The three-dimensional structure of A. pernix
CR was determined at a resolution of 2.09 Å. The overall
fold of A. pernix CR showed moderate similarity to thatof Tx chicken fatty liver CR; however, A. pernix CR had
no active-site lid unlike Tx chicken fatty liver CR.
Consequently, the active-site cavity in the A. pernix CR
was much more solvent-accessible than that in Tx
chicken fatty liver CR. This structural feature may be
responsible for the enzyme’s lower affinity for several
substrates and NADPH. The factors contributing to themuch higher thermostability of A. pernix CR were also
analyzed by comparing its structure with that of Tx
chicken fatty liver CR. This is the first report regarding
the characteristics and three-dimensional structure of
hyperthermophilic archaeal CR.

Fig.1 The dimeric structure of A. pernix CR. The helices and -strands are shown in green and orange,respectively. The adjacent subunit is shown in gray. The
dimerization loop is involved between the two subunits.
NADPH molecule (magenta) is shown as a stick model.
Oxygen and nitrogen atoms are shown in red and blue,respectively.

2 ExperimentSingle-wavelength (1.0 Å) data for A. pernix CR wascollected on the beamline AR-NE3A at the Photon
Factory. The data were processed using HKL2000 andthe CCP4 program suite.
3 Results and Discussion
The structure of the NADPH-bound A. pernix CR wasdetermined using molecular replacement (MR) method
and was refined at a resolution of 2.09 Å to a
crystallographic R-factor of 22.9% and a free R-factor of
24.2% [3]. The asymmetric unit consisted of one dimer
with a solvent content of 64.5%, which corresponds to a
Matthew’s coefficient of 3.47 Å3Da-1. The final structure
showed good geometry with no Ramachandran outliers,
and consisted of 477 amino acid residues, two NADPH
molecules and 244 water molecules (Fig. 1, 2). The
atomic coordinates and structure factor (code 5B1Y) havebeen deposited to the Protein Data Bank (PDB).

Fig.2 The substrate binding cavity structure of A. pernix
CR. The structure of A. pernix CR is shown in pink.
NADPH (magenta) and the disorder region (L197 andR205) are shown as stick models. The substrate-binding
pocket structure of A. pernix CR is indicated. Atoms arecolored as described for Fig. 1.
References[1] K. Yoneda et al., Acta Crystallographica Section F.
(2012) F68, 1568-1570.
[2] Y. Fukuda et al., FEBS J. (2015) 282, 3918-3928.
[3] Y. Fukuda et al., Enzyme Microb. Technol. (2016) 91,17-25.
* kyoneda@agri.u-tokai.ac.jp