Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

10C/2004G175

Conformational change in Aeropyrum pernix 5’-methylthioadenosine
phosphorylase under extremely high temperature conditions
Shuichiro GODA1,#, Yasuhiro SHIMIZU1,$, Harumi YOSHIDA1, Haruhiko SAKURABA1, Toshihisa
OHSHIMA1,$, Yuzuru HIRAGI*,2
1
The University of Tokushima, Tokushima 770-8506, Japan
2
Kansai Medical University, Hirakata, Osaka 573-1136, Japan#Present address: Nagasaki University, Nagasaki 852-8521, Japan$Present address: Kyushu University, Fukuoka, 812-8581, Japan

Materials and MethodsRecombinant MTAP was expressed in Escherichia coli.
The E. coli strain BL21(DE3) codon plus RIL wastransformed with pET11a/MTAP. The transformants
owere cultivated at 25 C in 200 ml of a medium containing
2.4 g of trypton, 4.8 g of yeast extract, 1 ml of glycerol,
2.5 g of K2HPO4, 0.76 g of KH2PO4, and 10 mg ofampicillin, until the optical density at 600 nm reached 0.6.
Induction was carried out by the addition of 1.0 mM
isopropyl-β-D-thiogalactopyranoside to the medium, andthen the cultivation was continued for 3 h. MTAP waspurified by a Butyl-Toyopearl chromatography. SAXS
measurements and analysis were described elsewhere [1,o2]. aMTAP was purified by autoclave treatment at 121 C,
20min followed by a Butyl-Toyopearl chromatography.
Results and Discussion
MTAP was expressed in E. coli and the subunit
molecular mass of recombinant MTAP was determined tobe 31 kDa by SDS-PAGE, while that of the autoclaved
recombinant enzyme (aMTAP) was 135 kDa. On theother hand, both the proteins exhibited similar mobility in
non-reducing SDS-PAGE, which corresponds to 135 kDa.
SAXS analyses showed that both the enzymes, MTAP andaMTAP form quaternary structure and the Rg,z values
were 36.5 Å (Fig. 1 (a) and (b)). No difference in shape of1Log J(Q)

0.95(a)

0.9
0.85
0.8
0.75
0.7
0.00E+00 5.00E-03 1.00E-02 1.50E-02 2.00E-02
Q*Q (Å-2 )1.4
1.2J(Q)*Q*Q

Introduction
To find out extremely thermostable proteins in
hyperthermophilic archaea, we screened soluble proteinsoeven after autoclave (121 C, 20 min) treatment. As theresult, some autoclave resistant proteins were found in thecell extract of the hyperthermophilic archaeon A. pernix.
Among them, we found a protein whose molecular mass
was estimated to be 135 kDa by SDS-PAGE and the Nterminal amino acid sequence was identical to that of theAPE1885 gene product. This protein has been annotatedas a putative 5’-methylthioadenosine phosphorylase(MTAP) via genome analysis. The subunit molecularmass was calculated to be 30.7 kDa from the predicted
amino acid sequence. This indicates that the MTAP forms
SDS-resistant oligomeric structure (aMTAP) by autoclave
treatment. To elucidate the difference in the structure
between MTAP and aMTAP, small-angle X-ray scattering(SAXS) measurements were carried out.

(b)

1.0
0.8
0.6
0.4
0.2
0.00

0.05

0.1

0.15

0.2

Q (Å-1 )Figure1. Guinier (a) and Kratky (b) plot of the MTAP
(black line) and aMTAP (red line).
overall molecule and in circular dichroism spectra wasobserved.
We changed the strain of E. coli from BL21(DE3)codon plus RIL to Rosetta-gami that promotes disulfide
bond formation in the cell. In the results, product from theRosetta-gami formed SDS-resistant oligomeric structure.
These results suggest that the conformational change
induced by autoclave may be related to the formation ofdisulfide bond in the protein.
References[1] S. Goda et al., PF activity reprots 252 (2002).
[2] Y. Nishikawa et al., PF activity reports 236 (2003).
* yhiragi@yahoo.co.jp

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