1. 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. ABS at 263 nm (mAu) 0 100 200 300 0 5 10 15 20 25 30 min P1 P2 A Mg Mn Cu Co Fe Zn n. d. 0.5 n. d. 2.1 n. d. Inductively Coupled Plasma-Atomic emission spectrometry (ICP-AES) analysis of Fur7 %contents P1 P2 Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities Absorbance at 225 nm (AU) (min) 05 15 20 10 05 15 20 10 0 2 1 0 2 1 1,6-DHN (A)GPP (B) Aromatic Substrate (P) PPi (Q)Geranylated compounds E EA EAB -EPQEQ 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 Kuzuyama 1 1 Biotechnology Research Center, The University of Tokyo, Japan, 2 Faculty 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. a N.P., no products. Although the true physiological substrate of NphB is still under investigation, significant Mg 2+ -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 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 MgCl 2, 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. Relative activity Effects of metal ions on prenylation of flaviolin by Fur7 0 20 40 60 80 0 min 60 P3, P4 B 20 40 References 1 kb ermE XbaI HindIII fur7 NruI XbaIHindIII Streptomyces origin tsr β-gal Amp r E.coli origin pWHM 860 Apr r -dfur7 Apr r Construction of fur7 gene disruptant sup of dfur7 broth100 ml Tris HCl (pH8.0)25 mM Fur7 0.1 mg / ml GPP0.2 mM 30˚C 2hours Addtion of Fur7 and GPP to supernatant of dfur7 broth 40.5 min Biosynthetic gene cluster of furaquinocin 1152345678161718192021 furaquinocin biosynthetic gene cluster 14131211109 Fur7 GPP 5 x malonyl CoA Fur1 Fur2, 3, 4, 6 furaquinocin A Geranylation of 5,7-dihydroxy 2-methoxy- 3-methylnaphthalene-1,4-dione Hepes (pH 7.5)50 mM 5,7-dihydroxy 2-methoxy- 3-methylnaphthalene-1,4-dione 0.2 mg Fur7 1 mg/ml GPP0.2 mM 30 ˚C 2hours Extract with EtoAC 5,7-dihydroxy 2-methoxy- 3-methylnaphthalene-1,4-dione Physiological substrate of Fur7 involved in furaquinocin biosynthesis Because 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 gene nph: naphtherpin biosynthetic gene fnq;: 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 Fur7 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.