Photon Factory Activity Report 2019 #37 (2020)
BL-5A, BL-17A, AR-NW12A, AR-NE3A/2019G546

Crystallographic analysis of the enzymes involved in regulation of CoA metabolism
1

Ayako YOSHIDA1, Keiichiro TAKASHIMA1, and Takeo TOMITA1,2*
Biotechnology Research Center, The Univ. of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657,
Japan
2
CRIIM, The Univ. of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

1 Introduction
Coenzyme A (CoA) is a very important coenzyme which
consists of adenosine diphosphate, pantothenate, and 2thioaminoethane moieties. CoA forms a thioester-bondbetween its thiol-group and acyl-group of various
compounds and acts as the acyl-carrier in the metabolic
pathways, such as TCA cycle, β-oxidation, biosynthesis offatty acids and amino acids, and degradation of aminoacids.
In addition to the role in the metabolic pathway, CoAderivatives, such as acetyl-CoA and succinyl-CoA, areshown to be involved in the acyl modification of proteinsin recent years. The comprehensive analysis using LCMS/MS discovered that a lot of proteins are acylated (forexample, acetylation and succinylation) even in bacteria.
Interestingly, the proteins involved in metabolism arereported to be often acylated, suggesting that the protein
acylation has some roles in metabolic regulation. We haveidentified over 200 proteins acetylated in thermophilic
bacterium, Thermus thermophilus HB27 [1].
In this study, we focused on the metabolic regulation in
the cells based on the acyl-CoA, which are the key
metabolites, and conducted the crystallographic analyses
to reveal the structural basis of the regulatory mechanism.
We have worked on several subjects involved in theregulatory mechanisms. Here, we describe about CoAtransferase (CoAT), which catalyzes the transfer of CoA
moiety among two acyl-CoAs, and acyl-CoA synthetases(ACSs), catalyzing the ATP-dependent acyl-CoA
production from CoA and short-chain fatty acids, from T.
thermophilus HB27.
2 Experiment
Purification of recombinant proteins
Since it was revealed that CoAT interacts with aregulatory protein in T. thermophilus, recombinant
proteins of CoAT and the regulatory protein were preparedby the expression system in Escherichia coli. Two proteins
with or without affinity tag were overexpressed in E. coli
BL21(DE3) and the expression was induced by theaddition of 1 mM of isopropyl-β-D-thiogalactopyranoside(IPTG). The cells were disrupted and the proteins were copurified or separately purified with heat treatment, affinitychromatography or anion-exchange chromatography, andgel-filtration chromatography.
T. thermophilus has four homologous genes encoding
ACS. We prepared recombinant protein of one ACS
homolog with affinity tag. ACS was overexpressed in E.
coli BL21-codon plus-(DE3) RIL and the expression wasinduced by the addition of 0.1 mM IPTG. The tagged ACS
was purified by heat treatment, affinity chromatography,
and subsequent gel filtration chromatography.
Crystallization
CoAT and its regulatory protein were concentrated andused for crystallization screening at 10 mg/ml, respectively.
CoAT co-purified with the regulatory protein was also used
for crystallization screening. Crystallization drops werecomposed of each protein, its substrate (analogs), CoA andbutyrate, and its cofactors. Purified protein of ACS wasused at 5 mg/ml for crystallization screening and its
crystallization drops were composed of each protein, CoA,ATP and acetate. Screening of crystallization condition
using Crystal Screen I and II, PEG-ION screen (HamptonResearch), Wizard classic I, II, and III, and Wizard
Precipitant synergy (Rigaku regents) was carried out withhanging drop vapor diffusion method. Optimization of thecrystallization condition was also conducted by modifying
the concentration of precipitant and pH of buffer used.
3 Results and Discussion
X-ray diffraction analyses of CoAT
There were several conditions in which CoAT or its
regulatory protein were crystallized. Several diffraction
datasets were obtained, and we could determine thestructure of CoAT and its regulatory protein at 2.6 Å and2.1 Å resolution, respectively by using molecularreplacement method. In addition, we could crystallize
CoAT co-purified with its regulatory protein in several
conditions and obtain some diffraction datasets. Now, weare trying to determine the structure.
X-ray diffraction analyses of ACS
There were several conditions in which ACS wascrystallized with or without substrates. Several diffraction
datasets were obtained. Now, we are trying to determine
the structure.
References[1] Yoshida A., et al. Extremophiles. 23, 377, (2019).
* uttomi@mail.ecc.u-tokyo.ac.jp