Photon Factory Activity Report 2019 #37 (2020)

BL-5A, 17A, NW12A/2018G047

Crystallographic analysis of enzymes involved in the biosynthesis of natural products
possessing complicated chemical structures
1Takeo TOMITA1,2 Masaya Kobayashi1 and Tomohisa KUZUYAMA1,2,*Biotechnology Research Center, The Univ. of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657,
Japan
2
CRIIM, The Univ. of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

1 Introduction
Carquinostatin A (CQS) is a carbazole alkaloid isolated
from an actinomycete Streptomyces exfoliates. CQS
exhibits antioxidant activity, and is thought to act asneuroprotective agent in cerebral ischemia. Therefore, it isexpected to be a lead compound for inhibition ofneurodegenerative diseases.
Recently, we identified gene cluster responsible for CQS
biosynthesis through heterologous production and genedeletion [1]. Biochemical characterization of seven CQS
biosynthetic gene products (CqsB1-7) established the total
biosynthetic pathway of CQS. We found that CqsB2
cyclizes an unstable biosynthetic intermediate with acylside chain to create carbazole intermediate,
precarquinostatin (Fig. 1).
In this project, we conducted crystallographic analysis ofCqsB2 to elucidate the mechanism of unprecedented
enzymatic reaction.

Crystallization conditions were screened by thehanging-drop vapor-diffusion method using Crystal Screen(Hampton Research) and the Wizard crystallization screen
series (Emerald Bio). The screenings were set up using 2µl drops consisting of 1 µl reservoir solution and 1 µl 10mg ml-1 CqsB2 solution with and without 2 mM
intermediate precarquinostatin.
3 Results and Discussion
We determined crystal structure of CqsB2 by the singlewavelength anomalous diffraction (SAD) method with aSeMet-substituted protein [1]. CqsB2 consists of Nterminal arm and C-terminal core domain, which adopts
the similar structure with TcmN aromatase/cyclase, andforms a homo-dimeric structure (Fig. 2). Precarquinostatinbinds at the active site (Fig. 3A). The docking model boundwith the substrate was created based on the crystalstructure (Fig. 3B). The CqsB2 structure and structurebased mutagenesis study provided mechanistic insights
into the reaction of CqsB2.

Fig. 1: The reaction catalyzed by CqsB2
2 ExperimentProtein expression and purification
E. coli BL21-CodonPlus (DE3)-RIL cell possessing
pHIS8-CqsB2, which is a plasmid for expression of CqsB2
fused with histidine tag at the N-terminal, was grown in 2x YT broth in the presence of kanamycin (50 µg/ml) andchloramphenicol (30 µg/ml) at 303 K. The gene expression
was induced by adding 0.1 mM IPTG and the culture wascontinued for additional 12-14 h. The samples werepurified with Ni2+-resin column chromatography, andSuperdex 200 gel filtration column chromatography. The
homogeneity over 95 % of the purified CLDS wereverified by SDS-PAGE.
E. coli B834 (DE3) was transformed with pHIS8-CqsB2
to express SeMet-CqsB2 protein. The transformant wasgrown in SeMet Core medium and the gene expression wasinduced by adding 0.1 mM IPTG. The protein was purified
with the same method with that of native CqsB2 protein.
Crystallization

Fig. 2: Crystal structure of CqsB2

Fig. 3: Active site structure of CqsB2. (A) Active sitebound with the CqsB2 product, precarquinostatin. (B)Docking model bound with the CqsB2 substrate.
References[1] M. Kobayashi et al., Angew. Chem. Int. Ed. Engle. 58,
13349 (2019).
* utkuz@mail.ecc.u-tokyo.ac.jp