Photon Factory Activity Report 2016 #34 (2017)

BL-5A, AR-NW12, AR-NE3A/2016R-13

Crystallographic analysis of enzymes involved in fosfomycin biosynthesis

Suhee CHO1, Tomohisa KUZUYAMA1, Takeo TOMITA1,*Biotechnology Research Center, the University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
1 Introduction
Fosfomycin is a natural phosphonate antibiotic
discovered in the broth from a soil bacterium
Streptomyces fradiae and shows broad-spectrum activities
against Gram-positive and Gram-negative bacteria.
Therefore, fosfomycin is widely used to treat acutecystitis.
Although fosfomycin has a simple structure, thebiosynthetic pathway has not been fully elucidated. [1] [2]To date, the pathway has been proposed to involve five
sequential reactions catalyzed by Fom1, Fom2, FomC,Fom3, and Fom4 enzymes. The initial biosynthetic
reactions
begin
witha
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)
byFomC
dehydrogenase. The radical-SAM enzyme, Fom3, hasbeen proposed to catalyze methylation of the C2 of HEP
to form 2-hydroxypropylphosphonate (HPP). 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.
2 Experiment
Preparation of CyTase domain of Fom1
CyTase domain of Fom1 was overexpressed in E. coli
BL21-Codon-Plus (DE3)-RIL as a host using 0.1 mM
isopropyl-β-D-1-thiogalactopyranoside(IPTG)
forinduction. From cell lysate prepared by sonication,
2+TK0280 was purified through heat treatment, Ni -NTA
affinity chromatography and Superdex 200 gel filtration
chromatography.

Crystallization
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.
3 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 collected dataset of X-ray diffraction. The attempts todetermine the structure by molecular replacement method
were unsuccessful. Therefore, we prepared SeMetsubstituted protein of CyTase domain of Fom1 andcrystallized. We collected the MAD dataset and are now
trying to determine the structure by MAD method.
References[1] Seto & Kuzuyama., Nat. Prod. Rep. 16, 589 (1999).
[2] Metcalf & van der Donk, Annu. Rev. Biochem. 78, 65(2009).
* uttomi@mail.ecc.u-tokyo.ac.jp