Photon Factory Activity Report 2018 #36 (2019)

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

Crystallographic analysis of enzymes involved in the biosynthesis of natural products
possessing complicated chemical structures
Takeo TOMITA1 Masaya Kobayashi1 and Tomohisa KUZUYAMA1,*1
Biotechnology Research Center,
The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan
1 Introduction
Terpenoids have been an important resource forbiologically active compounds because of their structural
diversity (Maimone & Baran, 2007, Sacchettini & Poulter,1997). Over the past decades, various studies have been
performed to identify the biosynthetic mechanism for theterpenoid complexity. The complexity of the terpenoid
skeleton is generated by the condensation of C5 isopreneunits and subsequent cyclization. Usually, thesecondensation and cyclization reactions are independently
catalyzed by isoprenyl diphosphate synthase (IDS) andcyclase, respectively.
Recently, we have identified and characterized CLDP
synthase (CLDS) from a soil bacterium Streptomyces sp.
CL190, which produces lavanducyanin, a phenazine with
an N-linked cyclolavandulyl structure. CLDS catalyzesboth the condensation of two molecules of C5
dimethylallyl diphosphate (DMAPP) and subsequent
cyclization to form CLDP and we have proposed a likely
reaction mechanism for CLDS. CLDS belongs to cisisoprenyltransferase. This enzyme family catalyzes thecondensation of DMAPP to form compounds with
polyprenyl chains. Among this family, undecaprenyl
diphosphate synthase (UDS) catalyzes the cis-prenyl chain
elongation onto trans, trans-farnesyl diphosphate (FPP) toproduce undecaprenyl diphosphate (UPP), which isindispensable for the biosynthesis of bacterial cell wall.
The crystal structure of the enzyme from Escherichia coli
and Micrococcus luteus were determined and the structural
basis of the condensation reaction is proposed. CLDS is
unique since CLDS catalyzes not only condensation but
also cyclization of the intermediate, while CLDS and UDS
share conserved amino acid residues that recognize thephosphate moiety of the prenyl diphosphate substrate.
In the present study, to gain insight into structural basis of
this unusual CLDS-catalyzed two step reaction, we
crystallized CLDS and then we collected and analyzed Xray diffraction data from the crystals of CLDS.
2 ExperimentProtein expression and purification
E. coli BL21-CodonPlus(DE3)-RIL cells possessing
pHIS8-CLDS-NH or pET-CLDS-CH, those are plasmids
for expression of CLDS fused with histidine tag at the Nor C- terminal, respectively, were grown in 2 x YT brothin 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 75 gel filtration column chromatography. The
homogeneity over 95 % of the purified CLDS wereverified by SDS-PAGE. CLDS-NH and CLDS-CH of over40 mg were purified.
Crystallization
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 10-1mg ml CLDS solution with and without 5 mM DMAPPand 2 mM MgSO4. We also performed screening using
CLDS solution containing 5 mM DMSPP, which is ananalog of DMAPP, and 2 mM MgSO4 to capture pseudopre-reaction state of CLDS.
3 Results and Discussion
We determined crystal structure of CLDS by the singlewavelength anomalous diffraction method with a SeMetsubstituted protein (Fig. 1) [1]. CLDS adopts typical foldfor cis-prenyl synthases and forms a homo-dimericstructure. PPi formed as a by-product of the CLDS reaction
and remained at the active site with Mg2+ ion and Tris
because we used the buffer containing Tris, DMAPP, andMgSO4 in the crystallization mixture. An in vitro reaction
using a regio-specifically 2H-substituted DMAPP
substrate revealed the intramolecular proton transfer
mechanism of the CLDS reaction. The CLDS structure andstructure-based mutagenesis provide mechanistic insights
into this unprecedented terpene synthase [1-2].

Fig. 1: Crystal structure of CLDS
References[1] T. Tomita et al., Angew. Chem. Int. Ed. Engle. 56,14913 (2017).
[2] M. Kobayashi et al., Chembiochem. 20, 29 (2019).
* utkuz@mail.ecc.u-tokyo.ac.jp