第 32 回 (平成 16 年度) 研究奨励金受領者研究報告

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32 16

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(7)

Nonribosomal peptide synthetase !"#$%

1. Echinomycin&'()*+,-./

Echinomycin

Streptomyces lasalien- sis

DNA !"#$%&'%()"

*(+,-./012345 6 789:2 2 ;<=>?)@A!"#$BCD5E F

*%"G(HI 40 kb D5:./0JKD2 6 LMNOPQRE primer unit ST. qunioxaline- 2-carboxylic acid (QC, 2)

ecm2, -3, -4, -8, -11, -12, -13,

-14 123./0JE U 1 VWXYZ

[$\]

ecm1, -6, -7

^_

fabC

123`E a b1

ecm17

^_

-18

123cde: thioacetal fg h0ij.0kl 6 /mDF%"G(1 nNo)pqE r 17 ;stklut: open reading frame (ORF) 0 1 ;stvw ORF ^_ 1 ; vxNG+yz)!(${ )@|S}

(Table 1) 6

2. QC01 QC'()234*5,$%

QC

2

~LMN}st1

E Scheme 1 1{ 1k{ 6 U

zST.

A

-tryptophan J adenylation E amino- acyl thioester WJ` bE hydroxyla- tion, pB1 3 h0ij. (Ecm2, -8, -12, -13) 6 }3&+$(E #$pB 1

b

-hydroxykynurenine (

4

) )q. (Ecm11, -14) 6

Table 1. Deduced function for the open reading frames of the echinomycin biosynthetic gene cluster.

ORF Amino Acids Sequence Homolog Putative Function

ecm1 527 Peptide arylation enzymeentE QC activation

ecm2 248 Type II thioesterasegrsT QC biosynthesis

ecm3 362 Isopropylmalate dehydrogenaseleuB QC biosynthesis

ecm4 472 FAD-dependent oxidoreductaseubiH QC biosynthesis

ecm5 n.a. Transposase (inactive) Unknown

ecm6 2608 Nonribosomal peptide synthetaseteiC Peptide synthesis modules 1῍2 ecm7 3135 Nonribosomal peptide synthetaseacmC Peptide synthesis modules 3῍4

ecm8 70 MbtH-like proteinmbtH Unknown

ecm9 181 DNA-binding response regulatorompR Regulation

ecm10 252 TetR family transcriptional regulatorpip Regulation

ecm11 220 Tryptophan 2,3-dioxygenasetdo2 QC biosynthesis

ecm12 395 Cytochrome P450 oxidasecypX QC biosynthesis

ecm13 598 Mannopeptimycin peptide synthetasemppB QC biosynthesis

ecm14 402 Erythromycin A esteraseereB QC biosynthesis

ecm15 285 Helix-turn-helix transcription regulatormarR Regulation

ecm16 806 Excinuclease ATPaseuvrA Self resistance

ecm17 313 Thioredoxin reductasetrxB Disulﬁde formation

ecm18 224 SAM-dependent methyltransferasesmtA Thioacetal formation fabC 82 Fatty acid synthase acyl carrier proteinacpP QC carrier protein n.a.: not applicable.

Scheme 1. Predicted pathway for quinoxaline-2-carboxylic acid biosynthesis.

(8)

5

6

!"

#$ %& QC '()* (Ecm3, -4) + ,-.

/0123456'( 71891:;<=4

>?*+

@> QC '(ABC

ecm2, -3, -4, -8, -11, -12, -13,

-14 1D,<=+ EFGH 7 '(AB CI J KL>MNOPQRSR 8 T)U1 '(ABC1D,VW+ @XYZ QC [\H ]^ 8 T1 QC '(ABCI J T7 promoter, ribosome binding site _%` T7 termina- tor a T 7 promoter %bcd>D,)*

multiple gene polycistronic D,MNOP A ef+

7 E>g.1 PKS _%` NRPS 1hij3 k I1g.GlZ1 GC m 1nh i>?7op + I7>7 '(AB CXYZ>nD,\HXYZ_qr stuvw9)

tRNA

tPux]^

D,yz{|} 7 ~\7o>'(A BCnD,\D,MNOP B +

3. Echinomycinoctadepsipeptide

echinomycin

Echinomycin '(1:;G Scheme 2

%+ NRPS 1 A uvon

) Ecm1 % QC adenylation

ArCP o aryl thioester 456()

*+ @> 2 T1 NRPS P M1, M2 >e(

* Ecm6 %

A

-Ser _%`

A

-Ala ' *+ 7 1 M1 G

A

-Ser

14)*

E uvmE *+ 2 T1 NRPS P

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A

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A

-Val @' *+ 71' M3, M4 G

N-

¡

>¬ 6_%`%&

8 *+

Ecm17 %&z®ua'( triostin A (7) ¡o * Ecm18 1

«>

S-

¡ sulfonium ylide thioa- cetal ( *o (Scheme 4) +

NRPS ¯ / H G PCP u v phosphopantetheinyl %&% holo *¥

?* XYZG71°±)*H

²1Z1ABC (sfp) °±)*7o+ E echinomycin '(ABCN³zOPG

Strepto- myces peucetius

daunorubicin '(ABC1´

oNµP¶v· ¸ABC

drrC

on

)

ecm16

VW+ XYZZ6¹>'(

echinomycin 1 DNA a'%*º»XYZ1¼

Scheme 2. Predicted pathway for echinomycin biosynthesis and the modular organization of the echinomycin NRPS.

ῌ12ῌ

(9)

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, -

16

, -

17

, -

18

,

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7

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4. QC

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fY Ni-NTA ghVijk)hlm7n o

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5. echinomycin echinomycin

triostin A

p q echinomycin ! " # $ % & 7 5

echinomycin (

1

) 678 triostin A (

7

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6

, -

7

, -

16

, -

17

, -

18

,

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BL21 (DE3) le῎W_῏

2 L &_῏;!"GH 0.3 mg ,ῑcE

Ht ESI-MS 7,%ΐ 1139 [M K]

1123 [M Na]

1101 [M H]

678 1053 [M-SCH3]

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1

H-NMR 7 1 F< F<= 16 2&!

"abt`5efY.u1 \ ]^7n 1 !G5~F< F;

thioacetal &X"F<

ecm18

r

4W triostin A !"#$%&En

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¹

H-NMR 7

7

F< n

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7

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Scheme 3. Proposed mechanism of dimerization and cyclorelease of the quinoxaline-tetrapeptide intermediates by the TE

domain.

Scheme 4. Proposed mechanism for the conversion of the disulﬁde bond to thioacetal bridge in echinomycin biosynthesis.

第 32 回 (平成 16 年度) 研究奨励金受領者研究報告

32 16

!"#$%&'()* + ,-./,012#3 DNA 456 789: ;<=>?@*

AB, CDEF;G-%HI!J KLE=@M#3 N@"#O(PQ$/412

#%R&STUV$WX SCFpof3 Y'5 Z() +*4* ;@AB,56[I!

PQ+,/412#3

%R& SCFpof3 () -\7#/ῌ SCFpof3 J ]J./@,23

SCFpof3 5Z01J2^* _

`a DNA (A 3@45 7#3

b67 c8dD DNA Ve9&f:H(g9+hi* (, 2:;j/b6J<klm#3 N6J=>Jnk o/pi12#q=< DNA ?rR@( SCFpof3

`9&8sHtpi,^,t* uABC4v wx* _`a DNA ?ryz%(D,{vwx/4 1|v}@M#/~!#3 ="#@JEF

412v SCFpof3 5Z DNA DGB

[  * HZ/I* J4* 5 6[v*Z/41? K#N/ +/

4v3

=PQ@*2vLM /#/* HU

Us 8 DNA 0N 2.5 mM / 5 m M.

Phleomycin  DNA O  P Q  DSB  15

g / ml.

TsA H$% V A: %H48VhN

50 mg/ml / 25  200 J/m

UV R VOSTU V3 [JWX2 30 10v3

SCFpof3 5ZL7#BJ ¡/

v3 Y 1 J HU / TsA 7#B * Y 2 J UV / DSB B/ ¢/v3

NN@* SCFpof3 5ZJ DNA Z[\£* DNA O

PQ* UV R* 2 L]^41* H

¤t9A2B ¥4v3 SCFpof3 J DNA DG

_`¦7# Rad Y'@J,23 }<<{§*

2 4B ¥4vN/t* abL

%&$¨©}+*@n#?(A2N/ ªc7

#3 /t{«* HU [/n*2v* ¬ H@

%f9H89:@}ª,­(d@nvN/t* e

fL g®89:4v2¯%(e^hWi

#E®%&$¨©DJi@M#3

v* SCFpof3 ( DNA OPQ41Jj9A 2B ¥7N/}{<0v3 NN/<* abL

 [7#/n* B°±t* DNA ²

",DG* _`(|v<³ /20v* L´x(

<fk@n#?(Mt* µcl23 iJ DNA DG¶,§* TsA /20v%H_m

 ·7#N/@_`a¸=¹nº» !#L

41}B ¥7 Y 1 ¡o N/t* 2L

 %&$¨©}i*?@M#3

=[wx* %R& SCFpof3 5Z *2* 5 6;,89:(@n#N/({<0v3 ¼a

rad

!"#$ #$%&'()*+!

,-$./0!1

2 345!-&$ 346$

7%&89:34; %!<751

="#:#:>$%?:34? &' (

20 )@AB!*+?:,-C DE.F D/F G-H&I0J!1 E. ( K DNA) -C

#$ L0/%12%J345#/67,

$ 'MN89O$2P5!345Q::;

%&1 21 )@AJ D/F /#R*S T<=?:&5UV1 >W 345?@GX

"!9:AY/9:ZBGC/,DEGR0W F GHI[345JKZ,!1 34 G LMS\]G,N^,J_OZB

(MOPAC) PJ QRS`a!'MN89Ob

C%GHAYcMdecfJ gZBDE Ohi345TUJKZH&1 "#%

V7%&jWjWk 5 l 7 X!Y% 9:AY DEOhijWJKZm,,G-!"

Z%&1 / ZBDET[n\5!&o jWk9pqrs (

) ZBGto0&

]^u%&1

vwexycz{jW (TrxR) NADPH _`%

O 5vwexyc ( J/ 10 kDa) z{5!

FAD |ajWG-! }b 1 ~1 cd TrxR ry rOef 2 gh)eNc*9O (SeCys)

I[)ejWG-!

1 jWAYAJz{

i/ NADPH FAD kjkrxl/0

 m    ( T C e f n o (

Gln

Gly

Cys

SeCys

SeCys

Gly) Cys

SeCys S

Se   z { % ) e NrOO5vwexycz{5!1 #pG-m / !&o SeCys Cys qP%& Cys jWk r<jW 0.1  !1 "0 Cys vw rO (pKa K 8.5) SeCys )eNrO (pKa K 5.5) #

$st%J&o/0J!1 g"G Cys498

,u-! Gln494 O'MN8 (His, Arg, Lys . . .) qP% Cys498 vwrOsvw5 0W Cys jWGjWkGR!&1

vwexycz{jW C efnox%

rvx

Acetyl

Ser

Ile

#%R&STUV$WX SCFpof3 Y'5 Z() +4 ;@AB,56[I!

`9&8sHtpi,^,t* uABC4v wx* _`a DNA ?ryz%(D,{vwx/4 1|v}@M#/~!#3 ="#@JEF

412v SCFpof3 5Z DNA DGB

[ * HZ/I* J4* 5 6[v*Z/41? K#N/ +/

=PQ@2vLM /#/ HU

Us8 DNA 0N 2.5 mM / 5 m M.

Phleomycin DNA O P Q DSB 15

TsA H$% V A: %H48VhN

50 mg/ml / 25 200 J/m

UV R VOSTU V3 [JWX2 30 10v3

SCFpof3 5ZL7#BJ ¡/

v3 Y 1 J HU / TsA 7#B * Y 2 J UV / DSB B/ ¢/v3

PQ* UV R* 2L]^41* H

¤t9A2B ¥4v3 SCFpof3 J DNA DG

_`¦7# Rad Y'@J,23 }<<{§*

24B ¥4vN/t* abL

#3 /t{«* HU [/n2v ¬H@

%f9H89:@}ª,(d@nvN/t* e

fL g®89:4v2¯%(e^hWi

v* SCFpof3 ( DNA OPQ41Jj9A 2B ¥7N/}{<0v3 NN/<* abL

[7#/n* B°±t* DNA ²

",DG* _`(|v<³ /20v* L´x(

<fk@n#?(Mt* µcl23 iJ DNA DG¶,§* TsA /20v%H_m

·7#N/@_`a¸=¹nº» !#L

41}B ¥7 Y 1 ¡o N/t* 2L

%&$¨©}i*?@M#3

=[wx* %R& SCFpof3 5Z 2 5 6;,89:(@n#N/({<0v3 ¼a

FAD |ajWG-! }b 1 ~1 cd TrxR ry rOef 2 gh)eNc*9O (SeCys)

1 jWAYAJz{

i/ NADPH FAD kjkrxl/0

m ( T C e f n o (

Se z { % ) e NrOO5vwexycz{5!1 #pG-m / !&o SeCys Cys qP%& Cys jWk r<jW 0.1 !1 "0 Cys vw rO (pKa K 8.5) SeCys )eNrO (pKa K 5.5) #

$st%J&o/0J!1 g"G Cys498

,u-! Gln494 O'MN8 (His, Arg, Lys . . .) qP% Cys498 vwrOsvw5 0W Cys jWGjWkGR!&1

vwexycz{jW C efnox%

rvx

Gly (end) Chem3D !Gy%&1 z@ Cys Cys 9 O SeCys Cys {|i5!1 "r

vx MM2 ZBGAA7}~9H

WinMOPAC }~ $¡¢G/9:ZBH

&1 C efgh Cys 9O,u-!

Gln 9O£'MN8qP%&rvxV

$ J?GX"! Cys Cys 9OsAY¤MrT' AM1, PM3, PM5 PJZB%&1 g¥¦ Q494R

§ Q494H ¨'r©9O§ªv9O 4

kcal/mol [Jk9p«r© (

) G Cys 9 Os/ !"DE/0& }b 2 ~1

His 9O*M¬r® Cys498 |¯VP,

W°[-!!*M¬r®±T

#!s s²³/0!G"0 ,%n\%& . "#n\wo!j G /0& His 9O 3 9O!-! Ser 9O His 9O´Rµ 0 g8O N

n¶#

$*M¬r®Oo0!,Z·/0

&1 g"G Ser 9O Ala 9OqP%& S491A/Q494 H *[JAYDEH&¥¦ 8O

¸ His ¹¦/ºo!DE/0&1 ZBPJ&¤MrT' AM1, PM3, PM5 jG AM1 H»%§5i O

G¼[UB! AY (IRC)

3 ~4 J"56 6 .

Q494H Q494R KHxT (L9

P7 Cys498 $9A

-QP 4 S491A-Q494H N 6.8 -

9R S RSM Ser491 ) H 494 wT UV-)79A- W-. 4 pH b3|rlL

-7 HxTMXY pH Z9W- .7 "U<(M6"HA"

9 f[-HxT Q494R, Q494H, S491A/Q 494H ,.\[K%|rl&T"U9A p3;9$ (

) 5 T"U9Ap3;

9$\ ] ^-

\_`L

6 abBcdDe" Q494R HxT,. Hx?0M X498C )7M9 Ap3;9$ (

) ;-.7 ]) ab\\ ^f g rg ?

jkRS"'

His Tab Arg T)7Ml "m no. ¡}¢£$¤~-.pdi 9¥ q,7

rs¥

Cys498 t¦>I Ser491 His T

&u av