Photon Factory Activity Report 2014 #32 (2015) B

NW12, NE3A, 5A/2014R10

Crystallographic analysis of enzymes involved in fosfomycin biosynthesis
Suhee CHO, Tomohisa KUZUYAMA, Takeo TOMITA*1
Biotechnology Research Center, the University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Introduction
Fosfomycin is a natural phosphonate antibiotic
discovered in the broth from a soil bacterium
Streptomyces fradiae (Hendlin, et al., 1969) and shows
broad-spectrum activities against Gram-positive andGram-negative bacteria (Allerberger & Klare, 1999;
Nakazawa, et al., 2003; Cassone, et al., 2004). Therefore,fosfomycin is widely used to treat acute cystitis.
Although fosfomycin has a simple structure (Christensen,et al., 1969), the biosynthetic pathway has not been fully
elucidated (Seto & Kuzuyama, 1999; Metcalf & van derDonk, 2009). To date, the pathway has been proposed toinvolve five sequential reactions catalyzed by Fom1,Fom2, FomC, Fom3, and Fom4 enzymes. The initial
biosynthetic reactions begin with a conversion ofphosphoenolpyruvate (PEP) to phosphonopyruvate. This
conversion is catalyzed by PEP phosphomutase, which isan enzyme common to the biosynthesis of all natural
phosphonates including the herbicide bialaphos and theantibiotic FR-900098. In fosfomycin biosynthesis, Fom1
catalyzes the first step to form phosphonopyruvate, which
is decarboxylated to phosphonoacetaldehyde byphosphonopyruvatedecarboxylase(Fom2).
Phosphonoacetaldehyde is then reduced to 2hydroxyethylphosphonate
(HEP)
by
FomC
dehydrogenase. The radical-SAM enzyme, Fom3, hasbeen proposed to catalyze methylation of the C2 of HEPto form 2-hydroxypropylphosphonate. Finally, 2hydroxypropylphosphonate epoxidase (Fom4) converts
HPP to fosfomycin.
Curiously, Fom1 has an extra cytidylyltransferase(CyTase) domain at the N-terminus in addition to the Cterminal PEP phosphomutase domain, whereas neither
BcpB nor FrbD encoding each PEP phosphomutase forthe biosynthesis of bialaphos and FR-900098 has theCyTase domain. Here, to obtain a clue to elucidate thefunction of CyTase domain, we expressed, purified,crystallized, and obtained preliminary X-ray diffraction
data of CyTase domain of Fom1 from a fosfomycinproducing Streptomyces wedmorensis.
Materials and Methods
Purification
Fom1(N) was overexpressed in E. coli BL21-CodonPlus (DE3)-RIL as a host using 0.1 mM isopropyl--D-1thiogalactopyranoside (IPTG) for induction. From celllysate prepared by sonication, TK0280 was purified
throughheat
treatment,
Ni2+-NTA
affinity
chromatography and Superdex 200 gel filtration
chromatography.

Crystalization
Screening of crystallization condition was initiallyperformed with Crystal screen I and II, Wizard classic I,
II, and III, and PEG/ION by hanging drop vapor diffusion
method. The crystallization conditions were further
optimized by changing pH and concentration ofprecipitant.
Data collection and processing.
The X-ray diffraction data of native proteins werecollected using the beamline, NW12 at PF.
The image sets were integrated and scaled using
HKL2000.
Results and Discussion
Expression, and purification of CyTase domain
Recombinant CyTase domain fused with histidine tag at
the N-terminal was expressed in E. coli and purified
through successive column chromatographies. CyTase
domain was purified to homogeneity on SDS-PAGE
analysis.
Crystallization and collection of datasets
As a result of crystal screening, we obtained a crystal. We
performed optimization the crystallization condition. We
are now trying to dataset of X-ray diffraction data at PF.
Further studies will provide the first structure of theCyTase domain of Fom1 involved in phosphonate
biosynthesis and also a clue to understand the mechanismof substrate recognition, which will clearly define thefunction of the CyTase domain in fosfomycin
biosynthesis.
* utkuz@mail.ecc.u-tokyo.ac.jp