Photon Factory Activity Report 2017 #35 (2018)

BL-5A, BL-17A, AR-NE3A, AR-NW12A/2016G162

Crystallographic analysis of enzymes involved in terpenoid biosynthesis
Takeo TOMITA, Masaya KOBAYASHI, and Tomohisa KUZUYAMA*
Biotechnology Research Center, The University of Tokyo, Tokyo 113-8657, Japan
1 Introduction
Terpenoids have been an important resource forbiologically active compounds because of their structural
diversity [1-2]. Over the past decades, various studies
have been performed to identify the biosynthetic
mechanism for the terpenoid complexity. The complexityof the terpenoid skeleton is generated by the condensationof C5 isoprene units and subsequent cyclization. Usually,
these condensation and cyclization reactions areindependently catalyzed by isoprenyl diphosphatesynthase (IDS) and cyclase, 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 catalyzes
both 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 withpolyprenyl chains. Among this family, undecaprenyl
diphosphate synthase (UDS) catalyzes the cis-prenyl
chain elongation onto trans, trans-farnesyl diphosphate(FPP) to produce undecaprenyl diphosphate (UPP), whichis indispensable for the biosynthesis of bacterial cell wall.
The crystal structure of the enzyme from Escherichia coli
and Micrococcus luteus were determined and thestructural 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 acidresidues that recognize the phosphate moiety of theprenyl diphosphate substrate.
In the present study, to gain insight into structural basisof this unusual CLDS-catalyzed two step reaction, wecrystallized CLDS and then we collected and analyzed Xray diffraction data from the crystals of CLDS.
2 Materials and Methods
Protein 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 theculture was continued for additional 12-14 h. The samples
were purified with Ni2+-resin column chromatography,
and Superdex 75 gel filtration column chromatography.
The homogeneity over 95 % of the purified CLDS wereverified by SDS-PAGE. CLDS-NH and CLDS-CH ofover 40 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). Thescreenings were set up using 2 µl drops consisting of 1 µl
reservoir solution and 1 µl 10 mg ml-1 CLDS solution
with and without 5 mM DMAPP and 2 mM MgSO4. We
also performed screening using CLDS solution containing
5 mM DMSPP, which is an analog of DMAPP, and 2 mM
MgSO4 to capture pseudo-pre-reaction state of CLDS.
3 Results and Discussion
Crystals of CLDS-NH appeared in a droplet containing
100 mM HEPES–NaOH pH 7.0, 24% PEG 2000 in thepresence of 5 mM DMAPP and 2 mM MgSO4. Crystalsof CLDS-CH appeared in a droplet containing 100 mM
Tris–HCl pH 7.0, 1 M sodium citrate tribasic, 200 mM
sodium chloride. X-ray diffraction data from these CLDS
crystals were collected and analyzed. CLDS-NH crystal
obtained in the presence of DMAPP belonged to spacegroup P1, with unit-cell parameters a = 46.9 Å, b = 61.7
Å, c = 82.2 Å, α = 74.0˚, β = 84.5˚, γ = 86.0˚. CLDS-CH
crystal obtained in the absence of DMAPP belonged tospace group P212121, with unit-cell parameters a = 39.0 Å,
b = 87.5 Å, c = 113.6 Å.
We determined crystal structure of CLDS by the singlewavelength anomalous diffraction method with a SeMetsubstituted protein [3]. CLDS adopts typical fold for cisprenyl synthases and forms a homo-dimeric structure. PPi
formed as a by-product of the CLDS reaction andremained at the active site with Mg2+ ion and Tris because
we used the buffer containing Tris, DMAPP, and MgSO4
in the crystallization mixture. An in vitro reaction using aregio-specifically 2H-substituted DMAPP substrate
revealed the intramolecular proton transfer mechanism ofthe CLDS reaction. The CLDS structure and structurebased mutagenesis provide mechanistic insights into thisunprecedented terpene synthase.
References[1] TJ. Maimone and PS. Baran, Nat Chem Biol. Jul;3(7),396 (2007)[2] JC. Sacchettini and CD. Poulter, Science, Sep 19;277
1788 (1997)[3] T. Tomita et al., Angew. Chem. Int. Ed. Engle. 56,14913 (2017).
* utkuz@mail.ecc.u-tokyo.ac.jp