QQQQ素 (LAO) Q L- Q脱Q素Q素 (L-aspDH) L- Q Q変Q 反Q Q媒

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NAD(P)

L- (LAO)!"# L-

$ (L-aspDH)%&#L-

'( )* +,1-.

/0'( 12(3 (QS)4 567DHAP89:# 2(3 +,1-.

'( 1;4<%0=# >?

42@A8BC4DEF. G0=#

LAO>L-aspDH1QS8H:7I8

EF7J. K0# NAD1# LM1L-N 3ONPQD45&/02(3:7RS . S0=# TN41LAO>QS1UVW# SFX 1YZ[\]^0=_`abcdN8`67 J. ef1ghi\%Pyrococcus horikoshii

<` J7QS jkl!D"4m:0

# # noCpq1$r4%%&# ECs bX&tXF7J`Juv%¹⁾. K0# c (qwNADx'y(z)*8:7# y(z { abH+=XF. y(z{ ab1

|45&}`# )4LAO!"L-aspDH8QS,2(

3~ ~3, (QPRTase)3y(z

y(z{ ab5-EF7J²⁾. SFX { ab`D"?X# ./\%Pyrobacu-

lum aerophilum1L-aspDH8QPRTasey(z0 :# EX4QSy(z8{ abH:7JS8

?60. SF1LAO8QS, QPRTase83 Ii:7J. #1QS, QPRTase3I81l!D"!"# LAO

+IlD"238:0.

P. aerophilumL-aspDH-QPRTase (PAE3114) QS (PAE3115)y(zGFF pET11a4{@b C:# G4\(BL-21-DE3 codon plus (DE3)) J 7u + 6 0. u 1 PAE3114- PAE31155y(zpET11a4{@bC:# 3 u60.

Sulfolobus tokodaii LAO (ST1196)y(z1

65&:77J0O ':

0. u14\BL-21-DE3 codon plus (DE3)

:# ¡41/¢8# 9$i{@£NP¤b J0. ¡ 22 mg/ml4:9:0# ¥:;

1 mM4`54FAD¦§# d¨¤@dO

©hª«<J# 20¬jk60.

P. aerophilumL-aspDH-QPRTase, QSy(

zpET11a4{@bCJ# SDS-PAGE45 u®d{60. L-aspDH-QPRTase (PAE3114)

¯K`JpET-11 a°{ab±N@b8:7

SDS-PAGEJ=²:0j³# >56 kDa&´4u a{+µ0 +,2,῏-.SF1# '(?@?

XEF¶·CdNzA(56426 Da)8¸B :0. :?:# pET-11a/PAE31141#CD4E

1. NAD(P)¹(

2. PAE3114SDS-PAGE45u®d{

M:zA£bob# ῌ: pET-11aE º# ῍: pET- 11a/PAE3114E º# ῎: ῌFG»# ῏:῍ FG»# ῐ: ῌw¼»# ῑ: ῍w¼»# ῒ: ῐ/¢8(90¬#10 min)w¼»# ΐ: ῑ/

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(2)

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012(34 5῎#6789:;<=

8 > L-aspDH-QPRTase, QS (PAE3114-PAE

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#67898 L-AspDH"#$%&'(

QS (PAE3115),OP$%&'(

S. tokodaii ,LAO (ST 1196), 3 Q Wizard II-3 (20RPEG8000, 0.2 M MgCl2, 0.1 M Tris/HCl bu#er pH 8.5),STI3Q U% V3Q1῏H9XWXY Z[12(34 \]^P212121_+`aab54.9AÕ, bb103.2 AÕ cb163.8 AÕ abbbgb90c ῎defΐ 43.9R, Matthew’s coe$cient2.2AÕ³/Da! g%'&(Native3Qhi1 mM-jklmn1 ompq8rs+FA,tmu1v`8w x y`hi ztn{|12H 2.09AÕῒA{}1y`

<=8(PDB IDb2E5V 3)³⁾ V3Q1῏

H-~,ompq1E 3Q NXWXYZ[5!( >,B M&

QK 16.5RPEG20000, 0.2 M CaCl₂, 0.1 M cacodylate bu#er pH 6.8 ,3Q1<8 L-~(0.5 M),ompq12H tmuv`12( >,34

\]^P31_+`aab136.6AÕbb136.6AÕcb70.3AÕ ab90cbb90cgb120ctmuv`<=8

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P. aerophilumῌῐL-aspDH-QPRTaseh

iQS)*+,H93?@A,<=

89H&HB wzkq)*+,12HK

,{|12῍`! S. tokodaiiLAO,FAD 3@67,{}<=8 !L-~

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1) H. sakuraba, H. Tsuge, K. Yoneda, N. Katunuma, and T.

Ohshima,J. Biol. Chem.,280, 26645῍26648 (2005).

2) K. Yoneda, R. Kawakami, Y. Tagashira, H. Sakuraba, S.

Goda, and T. Ohshima, Biochim Biophys Acta., 1764, 1087῍1093 (2006).

3) H. Sakuraba, K. Yoneda, I. Asai, H. Tsuge, N. Katunuma, and T. Ohshima,Biochim Biophys Acta.,1784, 563῍571 (2008).

3. S. tokodaiiῌῐLAO,OA{}

ῌ18ῌ