Photon Factory Activity Report 2017 #35 (2018)

BL-1A/2017G538

X-Ray Crystal Structure Analysis of Pseudoglycosyltransferase VldE
Chin Piow WONG1, Takahiro Mori2, and Ikuro Abe2,*
Institute of Natural Medicine, University of Toyama, Toyama, 930-0194, Japan
2
Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, 113-0033, Japan
1

3 Results and Discussion
The crystal structure of VldE D158N and H182A weresolved at 2.5 Å and 2.4 Å, respectively. The final R-value
was 24.2% (Rfree = 28.4%) for VldE D158N and 18.8%(Rfree = 24.0%) for VldE H182A. The mutants VldE
retains the wild-type homodimeric overall structure,
where each monomer consists of two Rossman 𝛽𝛼𝛽domains (Figure 2) [2] .

1 Introduction
VldE is a pseudoglycosyltransferase involved in thebiosynthesis of validamycin A, a naturally occurringtrehalose inhibitor that is used as antifungal agent toprotect crop [1]. VldE catalyzes a nonglycosidic C-Nbond formation between GDP-valienol and validamine 7phosphate to produce validoxylamine A 7’-phosphate(VDO) (Figure 1), an intermediate of validamycin A.
Previous mutagenesis experiment of VldE suggested that
Asp158 and His182 play the pivotal role in catalyzing theformation of VDO [2]. However, the mechanism
employed by VldE to catalyze the non-glycosidic C-Nbond is remained unclear. Hence, the crystal structures ofD158N and H182A mutants were elucidated in order tofurther clarify the underlying catalytic mechanism ofVldE.
OPO32-HOHN

H2NNOHHOHOOOOO P O P OOH
OO-GDP-valienolHOONNH2HOOHNvalidamine
7-phosphateHOOHVldEHOGDPOO P OOOHOHOHNHOHOH

validoxylamine A
7´-phosphate

Figure 1. Proposed reaction catalyzed by VldE.
2 Materials and Methods
Crystallization– Diffraction-quality
crystalsofVldE D158N were obtained after 2 days of incubation at20 ºC, in 100 mM Tris-HCl (pH 8.5) containing 26%
PEG4000, with 10 mg/mL of purified VldE D158N
solution. While the crystals of H182A were obtained
after a week on incubation at 4 ºC, in 100
mM Tris-HCl (pH 8.5) containing 26% PEG4000,with 10 mg/mL of purified VldE H182A
solution. Both crystals were obtained byusing sitting-drop vapor-diffusion method.
Data collection – Both type of crystals were transferred
into the soaking solution with 15% (v/v) ethyleneglycol
for 10 sec for cryoprotection and then flash cooled at-173°C in a nitrogen-gas stream. The X-ray diffractions ofcrystals were collected at BL-1A, processed andscaled with XDS. The structure was solved by thewith
Phaser-MR
molecular replacement
method(simpleone-component interface) using pseudoglycosytransferase (VldE, PDB accession code: 3VDN)
as search model. The structure was modified
manually with Coot and refined with PHENIX.

Figure 2. Overall structure of VldE H182A.
OHOH

The electron OHdensity map of D158N and H182A andthestructure of both mutants confirmed thatOH
HO elucidated
NOH H
Pi Asp158
wasOH successfully converted to Asn158, andvalidoxylamine A
His182to Ala182, respectively (Figure 3). The crystals
structures of these mutants complexed with the substrates,
GDP-valienol and validamine 7-phosphate, and theenzymatic product VDO will be prepared to fully
unraveled the importance of Asp158 and His182 for VldE
catalytic mechanism.

VldH

HOFigure 3. Close-up view on Asn158 and Ala182 residues.
D158N and H182A mutant is represented cyan and gold,respectively.
References[1] T. Iwasa, H. Yamamoto, M. Shibata, J. Antibiotic. 24,107-113 (1971)[2] M.C. Cavalier, Y.S. Yim, S. Asamizu, D. Neau, K.H.
Almabruk, M. Taifo, Y.H. Lee, PLOS ONE, e44934(2012)*abei@mol.f.u-tokyo.ac.jp