Photon Factory Activity Report 2016 #34 (2017)
BL-5A, BL-17A, AR-NE3A, AR-NW12A/2016G162

Crystallographic analysis of enzymes involved in terpenoid biosynthesis
Takeo TOMITA, Kenichi MATSUDA, Masaya KOBAYASHI, Taro OZAKI, Sarina TANAKA, andTomohisa 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 (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 (Ozaki et al., 2014).
CLDS catalyzes both the condensation of two moleculesofC5 dimethylallyldiphosphate(DMAPP)
andsubsequent cyclization to form CLDP and we haveproposed a likely reaction mechanism for CLDS. CLDSbelongs to cis-isoprenyltransferase. This enzyme family
catalyzes the condensation of DMAPP to form
compounds with polyprenyl chains. Among this family,
undecaprenyl diphosphate synthase (UDS) catalyzes thecis-prenyl chain elongation onto trans, trans-farnesyldiphosphate (FPP) to produce undecaprenyl diphosphate(UPP), which is indispensable for the biosynthesis ofbacterial cell wall. The crystal structures of the enzyme
from Escherichia coli and Micrococcus luteus weredetermined 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 the phosphate moietyof 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.

verified by SDS-PAGE. CLDS-NH and CLDS-CH ofover 40 mg were purified.
Crystallization
Crystallization conditions were screened by thehanging-drop vapour-diffusion method using CrystalScreen (Hampton Research) and the Wizard
crystallization screen series (Emerald Bio). Thescreenings were set up using 2 µl drops consisting of 1 µl
-1reservoir solution and 1 µl 10 mg ml 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 mMMgSO4 to capture pseudo-pre-reaction state of CLDS.
3 Results and Disccusion
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 = 61.7Å, 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. CLDS adopts typical fold for cisprenyl synthases and forms a homo-dimeric structure. PPiformed 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 MgSO4in the crystallization mixture. We designed the mutationof the active site and are now analyzing the characteristicsof mutant enzymes.

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 * utkuz@mail.ecc.u-tokyo.ac.jp
for expression of CLDS fused with histidine tag at the N- orC- terminal, respectively, were grown in 2 x YT broth inthepresenceofkanamycin(50µg/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 was