Photon Factory Activity Report 2022 #40 (2023)

BL-1A/2020G538

X-ray crystal structure analysis of fungal indole prenyltransferase withβ‐carbolines
Yu Nakashima1 and Hiroyuki MORITA1,*1
Institute of Natural Medicine, University of Toyama, Toyama 930-0194, Japan

1 Introduction
β-Carboline is a valuable indole alkaloid produced
by a variety of organisms, and it can be found inseveral food items, alcoholic beverages, and tobaccosmoke, as well as human tissues and body fluids.
Hence, we focused on studying the indole
prenyltransferase CdpNPT to introduce morediversity into β-carbolines. CdpNPT, originallyidentified in Aspergillus fumigatus and Neosartorya
fischeri, is highly substrate-tolerant and accepts
various compounds containing indole rings as prenylacceptors with dimethylallyl diphosphate (DMAPP)
as the prenyl donor to catalyze prenylation reactions[1, 2]. We have shown that CdpNPT can accept theβ-carbolines harmol and harman as prenyl acceptors,
forming corresponding prenylation products (Scheme1) [3, 4]. Therefore, in order to gain more insights into
the detailed mechanism behind these reactions, we
performed a crystal structure analysis of CdpNPT
complexed with both compounds.

3 Results and Discussion
The crystal structures of CdpNPT complexed withharmol and harman were solved by X-ray
crystallography at 2.40 Å and 2.43 Å resolution, withthe final R-value of 18.4% (Rfree = 22.3%) and 21.8%(Rfree = 23.6%), respectively. Harmol and harman
were observed in the reported typical prenyl
acceptor-binding region in the active-site cavity within
the central (ααββ)5 barrel and displayed several π
stacking interactions in the identical position (Fig. 1).
The catalytic Glu116 residue forms a hydrogen bondinteraction with the N-9 atom in the indole core of both
molecules, leading to the proposed prenylated
mechanism initiated by the proton abstraction of theindole amine by Glu116.

Scheme 1: Substrates and product of CdpNPT.
2 Experiment
Crystallization – Diffraction-quality crystals of theC-His6-tagged CdpNPT (38-440) with 2 mM harmol orharman were obtained at 20 ˚C, in 200 mM
ammonium phosphate dibasic and 20% (w/v)
PEG3350 (pH 8.0) with 10 mg/mL of purified CdpNPT
solution, by using sitting-drop vapor-diffusion
method.
Data collection – The crystals were transferred into
the soaking solution with 20% (v/v) glycerol for 10 secfor cryoprotection and then flash cooled at -173°C ina nitrogen-gas stream. The X-ray diffractions ofcrystals were collected at BL1A, processed andscaled with XDS and AIMLESS in the CCP4 program
package. The structures were solved by themolecular replacement method with Phaser-MR
using CdpNPT crystal structure (PDB entry 4E0U) asa template. The structures were modified manuallywith Coot and refined with PHENIX.

Fig. 1: Close-up view of the CdpNPT-harmol active
site (PDB code: 7XVJ) (A) and CdpNPT-harman
active site (PDB code: 7Y3V) (B), with arepresentative OMIT electron density map (mFoDFc) contoured to 4σ around harmol and 3σ aroundharman, respectively. The dashed lines represent thedistances in Å.

Photon Factory Activity Report 2022 #40 (2023)

Acknowledgement
We sincerely thank the PF staffs for their help withour data collections.
References[1] S-M. Li et al., Phytochemistry 70, 1746-1757(2009).
[2] H. Zou et al., J. Nat. Prod. 72, 44-52 (2009).
[3] S. A. Hamdy et al., J. Nat. Med. 76, 873-879(2022).
[4] S. A. Hamdy et al., J. Biosci. Bioeng. 134, 311-317(2022).
* hmorita@inm.u-toyama.ac.jp