Photon Factory Activity Report 2017 #35 (2018)

AR-NW12A, AR-NE3A, BL-5A, BL-17A/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 whichis consisted 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, 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 proteinacylation has some role in metabolic control via regulationof 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 several subjects involved in theregulatory mechanisms. Of which, here, we describe abouthomocitrate synthase (HCS), which catalyzes the first stepof lysine biosynthesis. This reaction is aldol-typecondensation of 2-oxoglutarate (2-OG) with acetyl-CoA toyield homocitrate. Previously analyzed bacterial HCS
consists of TIM barrel domain, subdomain I, andsubdomain II and is inhibited by lysine binding at 2-OGbinding site. On the other hand, archaeal HCS has aregulatory domain in addition to these three domains. We
hypothesized the regulatory domain was essential toregulate the activity of HCS and performed crystallizationof HCS. Here, we describe the recent progress of thisproject.
2 Materials and Methods
Purification of recombinant proteins
HCS 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 α-isopropyl-β-Dthiogalactopyranoside (IPTG) for induction. HCS wasdissolved in buffer A (20 mM Tris-HCl pH8.0, 150 mMNaCl) and purified with chromatography and thesubsequent gel filtration chromatography using Superdex
200.

Crystallization
HCS was concentrated and used for crystallization
screening at 5-10 mg/ml. Crystallization drops werecomposed of each protein and its (possible) substrates oran inhibitor, lysine. 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 and pH.
3 Results and Discussion
X-ray diffraction analyses of HCS
There was one condition from Precipitant synergy inwhich HCS was crystallized in the presence of 2-OG andacetyl-CoA. By optimization of this condition wesucceeded in obtaining better shape of crystals. However,only 6 Å resolution diffraction data from this crystal was
obtained and we cannot collect the diffraction data.
Optimizations of the crystallization condition and cryo
buffer will be necessary to obtain diffraction data sets.
* uttomi@mail.ecc.u-tokyo.ac.jp