Photon Factory Activity Report 2022 #40 (2023)

BL-1A, BL-5A, AR-NE3A/2021G555

Crystallographic analysis of the proteins involved in the CoA metabolism
1Ayako YOSHIDA1,2 and Takeo TOMITA1,2*
AgTECH, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2
CRIIM, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

1 Introduction
Coenzyme A (CoA) is an 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
and degradation of fatty acids and amino acids.
In addition to the role in the metabolic pathway, recently
CoA derivatives, such as acetyl-CoA and succinyl-CoA,are shown to be involved in the post-translational
modification of proteins like acetylation and succinylation.
The comprehensive analysis using LC-MS/MS discoveredthat a lot of proteins are acetylated even in bacteria.
Interestingly, the proteins involved in metabolism arereported to be often acetylated, suggesting that the protein
acetylation has some roles in metabolic regulation. It is
well-known that acetyl-CoA synthetase (ACS), which
produce acetyl-CoA in an ATP-dependent manner, isregulated by acetylation in a feedback manner. We haveidentified over 200 proteins acetylated in a thermophilic
bacterium, Thermus thermophilus HB27, and revealed thatan enzyme involved in the leucine biosynthesis is regulatedby the protein acetylation [1].
In this study, we focused on the metabolic regulation inthe 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 the regulation of ACS and CoAtransferase (CoAT), which was found as highly acetylated
protein in T. thermophilus. Here, we describe about CoAT,
catalyzing the transfer of CoA moiety of acyl-CoA to shortchain 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.

In addition, to maintain the native status of the protein
modification in CoAT, we prepared the complex of CoAT
and the regulatory protein using T. thermophilus strain
overproducing these proteins.
Crystallization
CoAT co-purified with the regulatory protein and CoAT
itself were used for crystallization screening.
Crystallization drops were composed of each protein, its
substrate (analogs), CoA and butyrate, and its cofactors.
Screening of crystallization condition using Crystal Screen
I and II, PEG-ION screen (Hampton Research), Wizard
classic I, II, III, and IV, and Wizard Precipitant synergy(Rigaku regents) was carried out with hanging drop vapor
diffusion method. Optimization of the crystallization
condition was also conducted by modifying theconcentration of precipitant and pH of buffer used.
3 Results and Discussion
X-ray diffraction analyses of CoAT
So far, we could determine the crystal structures of CoAT
at 2.6 Å but it does not contain any substrates. Therefore,
we tried co-crystallization with its substrates and crystal
soaking before flash freezing with liquid N 2. However, wecould not obtain the good diffraction from the crystals. We
suppose that it is necessary to find other crystallization
conditions or to crystallize homologous proteins fromother species.
X-ray diffraction analyses of the complex of CoAT and theregulatory protein
We have determined the structure of the regulatory
protein in the presence/absence of its ligand, but thecrystals from the screening using the mixture of CoAT andthe regulatory protein always contains only the regulatory
protein. Since the size of the complex is over 400 kDa, we
now try to determine the structure by CryoEM. Preliminary
analysis shows that those prepared as the complex of two
proteins contain a mixture of different ratios of CoAT andthe regulatory protein, which we believe madecrystallization difficult.
References[1] Yoshida A., et al. Extremophiles. 23, 377, (2019).
*uttomita@gmail.com