Photon Factory Activity Report 2017 #35 (2018)

BL-5A/2016G502, 2017G010

Crystal structure of the NADP+/tartrate complex of L-serine 3-dehydrogenase from thehyperthermophilic archaeon Pyrobaculum calidifontis
1

Kazunari Yoneda*1, Haruhiko Sakuraba2, Toshihisa Ohshima3
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 L-serine dehydrogenase (L-SerDH;EC 1.1.1.276) was identified in the hyperthermophilicarchaeon Pyrobaculum calidifontis JCM 11548/VA1.
The gene was overexpressed in Escherichia coli, and its
product was purified and characterized. The expressed
enzyme appears to be the most thermostable L-SerDH
described to date, and no loss of activity was observed byincubation for 30 min at temperatures up to 100C [1].
The enzyme showed substantial reactivity towards Dserine, in addition to L-serine. The crystal structures of P.
calidifontis L-SerDH were determined using themolecular replacement method: the structure in complex
with NADP+/L-(+)-tartaric acid (substrate analog) at 1.57
Å [2]. The fold of the catalytic domain showed highsimilarity with that of Ps. aeruginosa L-SerDH. However,
the active site structure significantly differed between thetwo enzymes. Based on the structure of the substrateanalog, tartrate, L- and D-serine molecules were modeled
into the active site and the substrate binding modes wereestimated. A structural comparison suggests that the widecavity at the substrate binding site is likely responsible forthe high reactivity of the enzyme toward both L- and Dserine enantiomers. This is the first description ofarchaeal L-SerDH structures with bound NADP+ andsubstrate analogue, and it provides new insight into thesubstrate binding mechanism of L-SerDH.

binding site via hydrophobic interactions. With theseinteractions, the tartrate molecule is tightly held near thenicotinamide ring of NADP+. Based on the structure of
the tartrate molecule, we modeled the L-serine (substrate)
molecule into the active site of P. calidifontis L-SerDH(Fig. 2A). In our predictive model, two oxygen atoms ofthe Cα carboxylate formed hydrogen bonds with the side
chains of Y218 and the carbonyl group of thenicotinamide ring, respectively. The oxygen atoms of theC OH group formed hydrogen bonds with the side
chains of T121, K170, and N173. As described above, P.
calidifontis L-SerDH exhibits substantial reactivity
toward D-serine (65% of the relative activity observedwith L-serine). We, therefore, modeled the D-serine(substrate) molecules into the active site of P. calidifontis
L-SerDH (Fig. 2B).

2 ExperimentSingle-wavelength (1.0 Å) data for P. calidifontis LSerDH was collected on the beamline BL-5A at thePhoton Factory.
The data were processed using
HKL2000 and the CCP4 program suite.

Fig. 1: The NADP +/tartrate-bound structure (5XVH).

3 Results and Discussion
In the electron density map of P. calidifontis LSerDH obtained from our experimental data, we noticed
an extra density within the active site cavity and foundthat a tartrate molecule could be modeled into that density
after construction and refinement of the peptide chain.
Indeed, the map clearly defined the precise orientation ofthe tartrate molecule (Fig. 1), and the oxygen atoms of theC1 and C4 carboxylates formed hydrogen bonds with theside chains of Y218 and T121. The oxygen atoms of theC2 and C3 OH groups formed hydrogen bonds with theside chains of Q174, K170, N173, and T121. In addition,
the active site lid structure (T206-G213) from anothersubunit appears to be involved in forming the substrate

Fig. 2: The NADP +/ L -serine-bound (A) andNADP +/ D -serine-bound structures (B).
References[1] K. Yoneda et al., Acta Crystallographica Section F.
(2013) F69, 134-136.
[2] K. Yoneda et al., Extremophiles (2018) 22, 395-405.
* kyoneda@agri.u-tokai.ac.jp