Photon Factory Activity Report 2014 #32 (2015) B

5A, NE3A, NW12A/2014G106

Crystallographic analysis of enzymes involved in terpenoid biosynthesis
Takeo TOMITA, Kenichi MATSUDA, Masaya KOBAYASHI, Taro OZAKI, Sarina TANAKA,
Makoto NISHIYAMA, and Tomohisa KUZUYAMA*
Biotechnology Research Center, The University of Tokyo, Tokyo 113-8657, Japan
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 moleculesof C5 dimethylallyl diphosphate (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 structure 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 basisof this unusual CLDS-catalyzed two step reaction, we
crystallized CLDS and then we collected and analyzed Xray diffraction data from the crystals of CLDS.
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 Pg/ml) andchloramphenicol (30 Pg/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 thepurified CLDS were verified by SDS-PAGE. CLDS-NHand CLDS-CH of over 40 mg were purified.
Crystallization
Crystallization conditions were screened by thehanging-drop vapour-diffusion method using Crystal
Screen (Hampton Research) and the Wizard
crystallization screen series (Emerald Bio). The screens
were set up using 2 Pl drops consisting of 1 Pl reservoir
solution and 1 Pl 10 mg ml-1 CLDS solution with andwithout 5 mM DMAPP and 2 mM MgSO4.
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, D= 74.0˚, E = 84.5˚, J = 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 attempted molecular replacement with UPPS fromCampylobacter jejuni (26% identity; PDB entry 3ugs;
Center for Structural Genomics of Infectious Diseases,unpublished work), UPPS from E. coli (27% identity;
PDB entry 1ueh; Chang et al., 2003) and UPPS from
Helicobacter pylori (27% identity; PDB entry 2d2r; Kuoet al., 2008) as search models. However, this attempt did
not provide a possible solution. Therefore, we are now
trying to determine the three-dimensional structure of
CLDS by the multiwavelength anomalous diffraction
method with a SeMet-substituted protein.

* utkuz@mail.ecc.u-tokyo.ac.jp