Photon Factory Activity Report 2022 #40 (2023)

BL-1A/2020G521

X-ray structure of lysoplasmalogen-specific phospholipase D
from Thermocrispum sp. RD004668
1Yoshiaki YASUTAKE1,2,*Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology(AIST), Sapporo 062-8517, Japan
2
Computational Bio Big-Data Open Innovation Laboratory, AIST, Tokyo 169-8555, Japan

1 Introduction
Lysoplasmalogen-specific phospholipase D (LyPlsPLD) from Thermocrispum sp. RD004668 is an enzymethat hydrolyzes choline lysoplasmalogen into choline and1-(1-alkenyl)-sn-glycero-3-phosphate.
Previousmutational studies revealed that the F211L altered its
substrate specificity from lysoplasmalogen to 1-OHexadecyl-2-hydroxy-sn-glycero-3-phosphocholine(lysoPAF) [1]. Enzymes specific for lysoPAF are rare andcould thus be used for clinical applications. In this study,
to understand the mechanism of substrate binding andselectivity, we have determined the crystal structure ofLyPls-PLD for the first time in substrate-free form [2].
2 Experiment
The tagless recombinant LyPls-PLD used in this study
was provided from Asahi Kasei Pharma Corp. The enzymesolution was prepared at 10 mg/mL using a buffer
containing 5 mM Tris-HCl pH 8.0 and 25 mM NaCl. The
LyPls-PLD was crystallized with sitting-drop vapor
diffusion method at 20 °C. Bipyramid-shaped crystals
were obtained using the reservoir solution containing 0.1
M Ches pH 9.5, 50 mM NaCl and 12% PEG 3350. The
crystals were transferred into a cryoprotectant solution
containing 0.1 M Ches pH 9.5, 200 mM NaCl, 18% PEG
3350 and 20% glycerol, and then flash-cooled in the liquid
nitrogen at 100 K. The X-ray diffraction data werecollected at PF BL-1A, and the data were processed using
the program XDS and AIMLESS from the CCP4 suite.
3 Results and Discussion
The structure of LyPls-PLD was determined by themolecular replacement method using the His-tagged
LyPls-PLD structure (PDB ID, 7YMP) as a search model[2]. The model refinement was performed up to aresolution of 2.9 Å, with R-factor and R-free factor of
17.8% and 21.5%, respectively (Table 1). The Asymmetricunit contains two LyPls-PLD monomers, which areextensively associated with each other by 2-foldsymmmetry. The strong electron density peak was detected
at the active site pocket, which was assigned as Ca2+ ion in
the course of model refinement. Overall structure of theLyPls-PLD represents a typical TIM barrel fold, and is
structurally
themosthomologous
totheglycerophosphodiester phosphodiesterase (GDPD) from
Thermoanaerobacter tengcongensis (Fig. 1). Based on thestructures obtained in this study, we expect to further

elucidate the substrate specificity swiching mechanism byusing molecular dymanics and docking simulatios.
Table 1: Data collection and refinement statistics
LyPls-PLD with Ca2+Space group
P43212
Unit cell (Å, deg)a = b = 142.95, c = 126.13
Resolution (Å)
2.91 (3.09-2.91)
Rmeas
0.111 (1.160)
I/σ16.1 (2.6)
Completeness (%)
100.0 (100.0)
Multiplicity
13.4 (14.3)
Rwork/Rfree
0.178/0.215

Fig. 1: Overall structure of LyPls-PLD. The bound Ca2+ is
depicted as a ball model colored in green.
Acknowledgement
The authors would like to thank the beamline staff at
the Photon Factory for their kind assistance during X-ray
diffraction experiments.
References[1] SI. Sakasegawa et al., Eur. J. Lipid Sci. Technol. 122,1900227 (2020).
[2] H. Hamana et al., Biosci. Biotechnol. Biochem. 87, 7481 (2023).
* y-yasutake@aist.go.jp