1. Kuzuyama T, et al. Nature. 2005, 435: 983-7.2. Kumano T, et al. Bioorg Med Chem. 2008, 16: 8117-26.3. Kumano T, et al. Jour. Biol. Chem. 2010 285: 39663-71.

OH

HO

HO OHO

O

OH

OH

HO O

O

HO

OH

OHO

OOH

HO

OH

O

O

HO

OH

OH

HO

OH

HO

HO

OH

OH

HO

HO OH

HO OH

O

O

OH

OH

HO

O

O

OH

OH

O

OHO

OH

OH

O

O

O

O

OH

OH O

OOH

O

OH

O

O

O

OH

OH

HO

OH

HO

OH

HOHO OH

O

O

OH

OH

HO

OH

HO

OH

HO

Nph

BS

CO

7 190

2-geranyl1,6-DHN

4-geranyl1,6-DHN

5-geranyl1,6-DHN

6-geranylnaringenin

7-O-geranylnaringenin 7-O-geranyl

apigenin

6-geranylapigenin

1,6-DHN 2,7-DHN olivetoldaidzeingenisteinapigeninnaringenin

N.P. N.P.

2

4

5

1

56

6 6

1

6

1

7

7 7

2

2

4

4

7

HO

OH

OH

HO

OH

OH

HO

OH

OH

resveratrol

2

4

1,3-DHN

Fur

7

N.P.a

N.P.N.P.

N.P. N.P. N.P. N.P. N.P. N.P.

OH

OH

OH

O

HO OH

HO

OH

OH

1-geranyl2,7-DHN

1,6-digeranyl2,7-DHN

7-O-geranylgenistein

7-O-geranyldaidzein

2-geranyl olivetol

4-geranyl olivetol

4-geranyl resveratrol

2-geranyl resveratrol1-geranyl 2,7-DHN

11

1-O-geranyl 1,3-DHN

2

2-dimethylallylresveratrol

4-dimethylallyl olivetol

2-dimethylallyl olivetol

6-dimethylallyl naringenin1-dimethylallyl 2,7-DHN5-dimethylallyl 1,6-DHN

O

O

HO

OH

8-geranyldaidzein

HO

OH

OH

C5H11

C5H11

2,4-di geranyl resveratrol

42

8

AB

S a

t 26

3 n

m

(mA

u)

0

100

200

300

0 5 10 15 20 25 30min

P1

P2

A

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

nometals

1 mMEDTA

MgCl2 MnCl2 FeCl3 CuCl2 NiCl2 CoSO4 CoCl2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

nometals

1 mMEDTA

MgCl2 MnCl2 FeCl3 CuCl2 NiCl2 CoSO4 CoCl2

MgMn

CuCoFe

Zn

n. d.0.5

n. d.n. d.2.1n. d.

Inductively Coupled Plasma-Atomic emission spectrometry (ICP-AES) analysis of Fur7

%contentsP1 P2

Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities

Abs

orba

nce

at 2

25 n

m (

AU

)

(min)0 5 15 2010 0 5 15 2010

0

2

1

0

2

1

1,6-DHN

1,6-DHN

B

1/[naringenin] (1/mM)-1 0 1 2

1/v

(min

/M

)

-1

0

1

2

3

4

5A

1/[GPP] (1/mM)-10 0 10 20

1/v

(min

/M

)

-1

0

1

2

3

4

5

(A)GPP(B) Aromatic Substrate

(P) PPi(Q)Geranylated compounds

E EA EAB -EPQ EQ E

A, (●) 0.5 mM, (○) 1.25 mM, (■) 2.5 mM, (□) 5 mM of naringenin and varied [GPP]B, (●) 0.05 mM, (○) 0.1 mM, (■) 0.2 mM, (□) 0.5 mM, (▲) 1 mM of GPP and varied [naringenin]

Natural products with one or more prenyl groups have been isolated to date mainly from higher plants. These compounds often posses various bioactivities. For example, prenylated flavonoids show promise as lead compounds for the development of novel pharmaceutical drugs. However, prenylated compounds are found at trace levels in natural sources and are not often amenable to synthesis in a cost effect manner.

Given the recent identification of catalytically promiscuous prenyltransferases displaying regiospecificity in prenyl group transfer and prenyl chain selectivity, these biocatalysts can serve as an alternate production strategy for natural product diversification and the chemo-enzymatic development of therapeutically novel synthetic compounds.

Aromatic prenyltransferase, which catalyzes the transfer of prenyl groups to an aromatic substrate, is a key enzyme in the biosynthesis of polyketide-terpenoid hybrid compounds such as the naphterpin, furaquinocin, napyradiomycin and BE-69785A.

Biosynthetic pathway of NphB and another polyketide-terpenoid hybrid compounds. They consist of polyketide moiety (blue) and terpenoid moiety (red).

Introduction

Results for NphB

○Takuto Kumano,1,2 Makoto Nishiyama,1 and Tomohisa Kuzuyama1

1Biotechnology Research Center, The University of Tokyo, Japan, 2Faculty of Lifi and Environmental Sciences, University of Tsukuba, Japan

We cloned and characterized the aromatic prenyltransferases NphB from Streptomyces sp. strain CL190, a naphterpin producer, SCO7190, a NphB homolog from S. coelicoler A3(2), Fur7 from Streptomyces sp. strain KO-3988, a furaquinocin producer, NapT8 and NapT9 from Streptomyces sp. strain CN-525, a napyradiomycin producer.

Here we report multiple syntheses of prenylated aromatic compounds by using prenyltransferases NphB, SCO7190, Fur7, NapT8 and NapT9, as biocatalysts.

aN.P., no products.

Although the true physiological substrate of NphB is still under investigation, significant Mg2+-dependent, in vitro activity is observed with 1,6-dihydroxy naphthalene (DHN).

(min)

　　 We solved the 3-D structures of NphB complexed with GPP alone and with GSPP and 1,6-DHN. However, we repeatedly failed to obtain NphB structures complexed with 1,6-DHN alone. This observation strongly suggests that 1,6-DHN cannot bind to NphB in the absence of GPP in the active site. GPP is thus most likely to be the first substrate to bind in the Sequential Ordered mechanism of the NphB reaction.

Lineweaver-Burk plots for the NphB reaction Antimicrobial activity of prenylated compounds

Results for Fur7

name of compoundsMIC value (µg/ml) againstStaphylococcus aureus

ampicillin 0.5apigenin >100daidzein >100genistein >100naringenin >100olivetol >100resveratrol >100

6-dimethylallyl naringenin 252-dimethylallyl olivetol 254-dimethylallyl olivetol 254-dimethylallyl resveratol 100

6-geranyl apigenin 256-geranyl naringenin 102-geranyl olivetol 54-geranyl olivetol 102-geranyl resveratrol 54-geranyl resveratrol 5

7-O -geranyl apigenin >1007-O -geranyl daidzein >1007-O -geranyl genistein >1007-O -geranyl naringenin >100

Fur7 has regular prenyltransferase activity and reverse prenyltransferase activity with GPP and DMAPP. However flaviolin was prenylated at C3. They are not intermediates of furaquinocin biosynthesis. So flaviolin is not a physiological substrate for Fur7.

An activity of no metals is 1.2U. Concentration of every metals are 1 mM.

No NphB

4-geranyl 1,6-DHN

5-geranyl 1,6-DHN

2-geranyl 1,6-DHN

(50 mM Tris-HCl (pH 8.0) ,5 mM flaviolin, 5 mM GPP. A, 5 mM DMAPP. B, 1 mg/ml Fur7),

HPLC; A:75%MeOH(0.1% AcOH), 2×150 mm ODS, B: 0-10min 40%, 10-50min 40-100%, 50-51min 100%MeOH (0.1% AcOH), 2×150 mm ODS

NphB -1,6DHN GPP

NphB, Fur7 and SCO7190 prenylated DHNs, flavonoids and plant polyketide.

7-O-geranylated flavonoids failed to exhibit anti-microbial activity, suggesting that 7-hydroxy group could be important for the activity.

Sequential mechanism

EA EAB

Fur7 shows the activity with both GPP and DMAPP

(50 mM Tris-HCl (pH 8.0) ,5 mM MgCl2, 5 mM 1,6-DHN, 5 mM GPP, 1 mg/ml NphB), HPLC; 80%methanol 4.6 x 250mm ODS

Fur7 -flaviolin GPP. A; DMAPP. B

Fur7 does not need metals for its activity. And Result of ICP-AES shows that there is no metals in Fur7.

Rel

ativ

e ac

tivity

Effects of metal ions on prenylation of flaviolin by Fur7

0 20 40 60 80

0

min

60 P3, P4B

20

40

References

HO

OH O

O

OHO

OH O

O

O

1 kb

ermE

XbaI HindIIIfur7

NruI NruI

XbaI HindIII

Streptomyces origin

tsr

β-gal

Ampr

E.coli origin

pWHM 860 Aprr-dfur7

Aprr

Construction of fur7 gene disruptant

sup of dfur7 broth 100 mlTris HCl (pH8.0) 25 mMFur7 0.1 mg / mlGPP 0.2 mM

30˚C 2hours

Addtion of Fur7 and GPP to supernatant of dfur7 broth

40.5 min

Biosynthetic gene cluster of furaquinocin

1 152 3 4 5 6 7 8 16 17 18 19 20 21

furaquinocin biosynthetic gene cluster14131211109

Fur7

GPP

HO

OH OH

OH

5 x malonyl CoA

Fur1

Fur2, 3, 4, 6

HO

O

O

O

O

HO

HO

furaquinocin A

Geranylation of 5,7-dihydroxy 2-methoxy-3-methylnaphthalene-1,4-dione

Hepes (pH 7.5) 50 mM5,7-dihydroxy 2-methoxy-3-methylnaphthalene-1,4-dione

0.2 mgFur7 1 mg/mlGPP 0.2 mM

30 ˚C 2hours

Extract with EtoAC

HO

OH O

O

O

5,7-dihydroxy 2-methoxy-3-methylnaphthalene-1,4-dione

HO

OH O

O

O

Physiological substrate of Fur7 involved in furaquinocin biosynthesisBecause that flaviolin is not a physiological substrate for Fur7, we try to find it from the fur7 disruptant strain.

Transform Streptomyces albus

fur: furaquinocin biosynthetic genenph: naphtherpin biosynthetic genefnq;: furanonaphthoquinone biosynthetic gene

6-(3,7-dimethylocta-1,6-dien-3-yl)5,7-dihydroxy 2-methoxy-3-methylnaphthalene-1,4-dione

TSB medium (30 μg/ml thiostrepton )

Pre-culture (30˚C, 2days)

Culture (27˚C, 3days)NMMP medium (30 μg/ml thiostrepton )

Centrifuge

Supernatant is used to Fur7 reaction.

Compound A

Compound B

A

A

A

B

B

NphBOHOH

HO OH

THN

OP

OHO

OP

O

HO

HO

GPP

OHPOH

O

OPO

HO

HO

O

H

HO

O

HO

OH

Naphterpin

O

O

HO

OH

Flaviolin

O2

Mg2+

PPi

OH

CoAS OH

O O

malonyl CoA

OCH3HO

O

O

O

HO

Furaquinocin

(Streptomyces sp. strain CL190)

(Streptomyces sp. strain KO-3988)

O

O

OOH

HO

ClCl

napyradiomycin(Streptomyces sp. CNQ-525)

Furanonaphthoquinone(Streptomyces cinnamonensis DSM 1042)

THN synthase

OCH3

O

OOH

O

HO

OH O

O

OH HO

OH O

O

OH HO

OH O

O

OHFur7

GPP

[O]

sup of dfur7 broth

Kuzuyama T, et al. Nature. 2005, 435: 983-7.

Kumano T, et al. Bioorg Med Chem. 2008, 16: 8117-26.

Kumano T, et al. Jour. Biol. Chem. 2010 285: 39663-71.