1.kuzuyama t, et al. nature. 2005, 435: 983-7. 2. kumano t, et al. bioorg med chem. 2008, 16:...
TRANSCRIPT
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.