Photon Factory Activity Report 2016 #34 (2017)

BL-5A, BL-17A, AR-NW12A, AR-NE3A /2015G546

Crystallographic analysis of the enzymes involved in regulation of CoA
metabolism
Tomohiro SUZUKI, Ayako YOSHIDA, and Takeo TOMITA*
Biotechnology Research Center, The Univ. of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
1 Introduction
Coenzyme A (CoA) is a very important coenzyme
which is consisted of adenosine diphosphate,pantothenate, and 2-thioaminoethane moieties. CoAforms a thioester-bond between its thiol-group andacyl-group of various compounds and acts as theacyl-carrier in the metabolic pathways, such as TCA cycle,β-oxidation, biosynthesis of fatty 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
LC-MS/MS discovered that a lot of proteins are acylated(for example, acetylated and succinylated) even inbacteria. Interestingly, the proteins involved inmetabolism are reported to be often acylated, suggestingthat the protein acylation has some role in metabolic
control via regulation of metabolic enzymes. Therefore,
the importance of CoA metabolism is increasing.
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 mainly two subjects; homocitratesynthase (HCS) from Sulfolobus acidocaldarius MW001,
which catalyzes the first step of lysine biosynthesis using
acetyl-CoA as a substrate, and CoA transferase (CoAT),
which is acetylated in T. thermophilus HB27. Here, we
describe the recent progress of these projects.

2 Materials and Methods
Catalytic domain of HCS
HCS from Sulfolobus acidocaldarius is composed ofthree domains: The N-terminal catalytic domain, theC-terminal regulatory domain (RAM domain), and thesubdomain I, and II connecting those two domains. We
designed expression system of the truncate body lacking
the C-terminal regulatory domain (HCS-cat).
Purification of recombinant proteins
HCS-cat with His-tag was overexpressed in Escherichia
coli BL21 (DE3) RIL as a host using 0.1 mM(IPTG)
for
α-isopropyl-β-D-thiogalactopyranoside
2+induction. HCS was purified with Ni -NTA affinity
chromatography and the subsequent gel filtration
chromatography using Superdex 200.

Crystallization
HCS-cat was concentrated and used for crystallization
screening at 4 mg/ml. Crystallization drops werecomposed of each protein and its (possible) substrates.
Screening of crystallization condition using Crystal
Screen I and II, PEG-ION screen (Hampton Research),and Wizard classic I, II, and III (Rigaku regents) was
carried out with hanging drop vapor diffusion method.
3 Results and Discussion
X-ray diffraction analyses of HCS-cat
There was one condition from Crystal Screen I in which
HCS-cat was crystallized in the presence of 2-oxogultarate
and acetyl-CoA. We obtained a diffraction data set fromthis crystal at 1.98 Å resolution. We are now trying todetermine the crystal structure by molecular replacement
method.
* uttomi@mail.ecc.u-tokyo.ac.jp