Construction of fur7 gene disruptant. Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities. Streptomyces origin. E.coli origin. pWHM 860 Apr r -d fur 7.
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Construction of fur7 gene disruptant Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities Streptomyces origin E.coli origin pWHM 860 Aprr-dfur7 ○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 Ampr Aprr tsr Introduction Results for Fur7 β-gal Kuzuyama T, et al. Nature. 2005, 435: 983-7. 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. Fur7 -flaviolin GPP. A; DMAPP. B XbaI HindIII ermE (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 300 XbaI HindIII B A fur7 P1 60 P3, P4 P2 200 1 kb NruI NruI 40 ABS at 263 nm (mAu) 100 20 0 0 0 10 20 5 15 30 25 0 20 40 80 60 min min Fur7 shows the activity with both GPP and DMAPP Biosynthetic pathway of NphB and another polyketide-terpenoid hybrid compounds. They consist of polyketide moiety (blue) and terpenoid moiety (red). 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. 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. Inductively Coupled Plasma-Atomic emission spectrometry (ICP-AES) analysis of Fur7 Effects of metal ions on prenylation of flaviolin by Fur7 Results for NphB 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). P1 P2 contents % Relative activity Mg 0.5 Mn n. d. NphB -1,6DHN GPP Fe n. d. Co n. d. (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 Cu 2.1 n. d. Zn 1,6-DHN No NphB 5-geranyl 1,6-DHN Fur7 does not need metals for its activity. And Result of ICP-AES shows that there is no metals in Fur7. 2 2 An activity of no metals is 1.2U. Concentration of every metals are 1 mM. 2-geranyl 1,6-DHN Physiological substrate of Fur7 involved in furaquinocin biosynthesis Absorbance at 225 nm (AU) 1 1 1,6-DHN Because that flaviolin is not a physiological substrate for Fur7, we try to find it from the fur7 disruptant strain. 4-geranyl 1,6-DHN Biosynthetic gene cluster of furaquinocin 0 0 furaquinocin biosynthetic gene cluster 10 15 0 5 20 10 15 0 5 20 (min) (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 NphB, Fur7 and SCO7190 prenylated DHNs, flavonoids and plant polyketide. fur: furaquinocin biosynthetic gene nph: naphtherpin biosynthetic gene fnq;: furanonaphthoquinone biosynthetic gene Transform Streptomyces albus Addtion of Fur7 and GPP tosupernatant of dfur7 broth Geranylation of 5,7-dihydroxy 2-methoxy- 3-methylnaphthalene-1,4-dione aN.P., no products. Kumano T, et al. Bioorg Med Chem. 2008, 16: 8117-26. sup of dfur7 broth A Lineweaver-Burk plots for the NphB reaction Pre-culture (30˚C, 2days) Antimicrobial activity of prenylated compounds TSB medium (30 μg/ml thiostrepton ) Hepes (pH 7.5) 50 mM 5,7-dihydroxy 2-methoxy- 3-methylnaphthalene-1,4-dione 0.2 mg Fur7 1 mg/ml GPP 0.2 mM 30 ˚C 2hours Extract with EtoAC Culture (27˚C, 3days) Compound A sup of dfur7 broth 100 ml Tris HCl (pH8.0) 25 mM Fur7 0.1 mg / ml GPP 0.2 mM NMMP medium (30 μg/ml thiostrepton ) 5,7-dihydroxy 2-methoxy- 3-methylnaphthalene-1,4-dione 30˚C 2hours A B Centrifuge Supernatant is used to Fur7 reaction. 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] 40.5 min A B Sequential mechanism (B) Aromatic Substrate (Q)Geranylated compounds (P) PPi • GPP Compound B 6-(3,7-dimethylocta-1,6-dien-3-yl) 5,7-dihydroxy 2-methoxy- 3-methylnaphthalene-1,4-dione EAB -EPQ EQ EA E E EAB EA 5 x malonyl CoA Kumano T, et al. Jour. Biol. Chem. 2010 285: 39663-71. Fur1 [O] Fur7 7-O-geranylated flavonoids failed to exhibit anti-microbial activity, suggesting that 7-hydroxy group could be important for the activity. GPP 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. Fur2, 3, 4, 6 Fur7 GPP furaquinocin A References 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.