Photon Factory Activity Report 2018 #36 (2019)

BL-5A, BL-17A, AR-NW12A, AR-NE3A/2018G573

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

Ayako YOSHIDA1 and Takeo TOMITA1*
Biotechnology Research Center, 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 metabolicpathways, such as TCA cycle, β-oxidation, biosynthesis offatty acids and amino acids, degradation of amino acids.
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, acetylated and succinylated) 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. One of theidentified acetylated proteins, 2-isopropylmalate synthase(IPMS), which catalyzes the first step of leucine
biosynthesis using acetyl-CoA as a substrate, was found tobe negatively regulated by protein acetylation [1].
In this study, we focused on the metabolic regulation in
the cells based on the CoA derivatives, 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 IPMS,
which is regulated by protein acetylation, and CoAtransferase (CoAT), which catalyzes the transfer of CoA
moiety among two acyl-CoAs, from T. thermophilus HB27.
2 Experiment
Purification of recombinant proteins
IPMS with affinity tag was overexpressed in Escherichia
coli BL21-codon plus-(DE3) RIL as a host. The expression
was induced by the addition of 0.1 mM Isopropyl-β-Dthiogalactopyranoside (IPTG) for induction. IPMS wasdissolved in buffer A (20 mM Tris-HCl pH8.0, 150 mMNaCl) and purified with affinity-chromatography and thesubsequent gel filtration chromatography using Superdex
200. CoAT with or without affinity tag was also
overexpressed in E. coli BL21-codon plus-(DE3) RIL andthe expression was induced by the addition of 1 mM IPTG.
The tagged CoAT was purified in the same way with IPMS.
The non-tagged CoAT was purified by in combination withhydrophobic interaction chromatography, anion exchange
chromatography, and gel filtration chromatography.

Purification of IPMS from T. thermophilus
IPMS was also prepared from T. thermophilus. We
constructed over-expressing strain of strep-tagged IPMS inT. thermophilus by using the constitutively expressingpromoter. The protein was purified with Strep-tactin
column, and Superdex 200.
Crystallization
IPMS purified from E. coli and T. thermophilus wereconcentrated and used for crystallization screening at 5-10
mg/ml, respectively. Crystallization drops were composedof each protein and its substrates or an inhibitor, leucine.
For CoAT, crystallization drops were composed of eachprotein, CoA, and butyrate. Screening of crystallization
condition using Crystal Screen I and II, PEG-ION screen(Hampton Research), Wizard classic I, II, and III, andWizard Precipitant synergy (Rigaku regents) was carriedout with hanging drop vapor diffusion method.
Optimization of the crystallization condition was also
conducted by modifying the concentration and pH.
3 Results and Discussion
X-ray diffraction analyses of IPMS
There was one condition from Crystal screen I in which
IPMS was crystallized in the presence of leucine. By
optimization of this condition, we succeeded in obtaininglarger crystals. However, only 3 Å resolution diffraction
data from this crystal was obtained and we cannot collectthe diffraction data. Optimizations of the crystallization
condition and cryo buffer will be necessary to obtaindiffraction data sets.
X-ray diffraction analyses of CoAT
There were several conditions in which CoAT wascrystallized with or without substrates. The diffraction
pattern for one of the crystals showed the crystal wasbelonged to the space group of primitive orthorhombic.
Although one data set was obtained at 2.7 Å resolution, it
was estimated as twinned. Further screening ofcrystallization conditions will be needed.
References[1] Yoshida A., et al. Extremophiles. 23, 377, (2019).
* uttomi@mail.ecc.u-tokyo.ac.jp