Binding of Two Flaviolin Substrate Molecules, Oxidative Coupling, and Crystal Structure of Streptomyces coelicolor A3(2) Cytochrome P450 158A2

Abstract

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 Å) and flaviolin-bound (1.62 Å) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers. Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 Å) and flaviolin-bound (1.62 Å) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers. Cytochrome P450 (P450 or CYP) 1The abbreviations used are: P450 or CYP, cytochrome P450 monooxygenase; THN, 1,3,6,8-tetrahydroxynaphthalene; CYP158A2, cytochrome P450 158A2; SRS, predicted substrate recognition sequences originally suggested in P450 sequences by Gotoh; COSY, correlated spectroscopy; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond coherence; HPLC, high pressure liquid chromatography. monooxygenases constitute a complex superfamily of proteins found in all biological kingdoms from bacteria to humans and participate in the biosynthesis of physiologically important compounds as well as in detoxification of a wide variety of foreign compounds from the environment (1Nelson D.R. Koymans L. Kamataki T. Stegeman J.J. Feyereisen R. Waxman D.J. Waterman M.R. Gotoh O. Coon M.J. Estabrook R.W. Gunsalus I.C. Nebert D.W. Pharmacogenetics. 1996; 6: 1-42Crossref PubMed Scopus (2663) Google Scholar, 2Porter T.D. Coon M.J. FASEB J. 1992; 6: 669-673Crossref PubMed Scopus (194) Google Scholar, 3Anzenbacher P. Anzenbacherova E. Cell. Mol. Life Sci. 2001; 58: 737-747Crossref PubMed Scopus (719) Google Scholar). Largely as a result of genome sequencing projects, >4000 genes within the P450 superfamily have been identified (drnelson.utmem.edu/CytochromeP450.html). Although there has been considerable progress in the expression and characterization of recombinant P450s within the last decade, the endogenous substrates of most remain unknown. For example, there are 18 CYP genes in the model actinomycete Streptomyces coelicolor A3(2) (4Bentey S.D. Chater K.F. Cerdeno-Tarraga A.M. Challis G.L. Thomson N.R. James K.D. Harris D.E. Quail M.A. Kieser H. Harper D. Bateman A. Brown S. Chandra G. Chen C.W. Collins M. Cronin A. Fraser A. Goble A. Hidalgo J. Hornsby T. Howarth S. Huang C.H. Kieser T. Larke L. Murphy L. Oliver K. O'Neil S. Rabbiowitsch E. Rajandream M.A. Rutherford K. Rutter S. Seeger K. Saunders D. Sharp S. Squares R. Squares S. Taylor K. Warren T. Wietzorrek A. Woodward J. Barrell B.G. Parkhill J. Hopwood D.A. Nature. 2002; 417: 141-147Crossref PubMed Scopus (2600) Google Scholar, 5Lamb D.C. Skaug T. Song H-L. Jackson C.J. Podust L.M. Waterman M.R. Kell D.B. Kelly D.E. Kelly S.L. J. Biol. Chem. 2002; 277: 24000-24005Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) whose endogenous functions and biological roles are largely unclear. S. coelicolor produces >20 secondary metabolites including antibiotics, siderophores, lipids, and pigments (5Lamb D.C. Skaug T. Song H-L. Jackson C.J. Podust L.M. Waterman M.R. Kell D.B. Kelly D.E. Kelly S.L. J. Biol. Chem. 2002; 277: 24000-24005Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Many of these compounds are oxidized and contain hydroxyl groups, which could arise from the function of P450s associated with the biosynthetic pathways. Most P450s catalyze monooxygenase reactions in which one atom of molecular oxygen is used to hydroxylate the substrate and the other is used to produce water (6Guengerich F.P. Crit. Rev. Biochem. Mol. Biol. 1990; 25: 97-153Crossref PubMed Scopus (256) Google Scholar). P450s additionally catalyze a myriad of other reactions, including epoxidations, oxidative rearrangements, and oxidative coupling reactions (7Guengerich F.P. J. Biol. Chem. 1991; 266: 10019-10022Abstract Full Text PDF PubMed Google Scholar, 8Brash A.R. Baertschi S.W. Ingram C.D. Harris T.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3382-3386Crossref PubMed Scopus (110) Google Scholar, 9Brash A.R. Baertschi S.W. Harris T.M. J. Biol. Chem. 1990; 265: 6705-6712Abstract Full Text PDF PubMed Google Scholar, 10Zenk M.H. Gerardy R. Stadler R. J. Chem. Soc. Chem. 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CYP158A2 from S. coelicolor A3(2) has also been implicated in phenol oxidative coupling (18Cortes J. Velasco J. Foster G. Blackaby A.P. Rudd B.A. Wilkinson B. Mol. Microbiol. 2002; 44: 1213-1224Crossref PubMed Scopus (58) Google Scholar), generating red-brown pigments in actinomycetes. The gene encoding this P450 (sco1207) is located in the three-gene operon sco1206-sco1208 that also contains the type III polyketide synthase 1,3,6,8-tetrahydroxynaphthalene (THN) synthase (19Austin M.A. Izumikawa M. Bowman M.E. Udwary D.W. Ferrer J.-L. Moore B.S. Noel J.P. J. Biol. Chem. 2004; 279: 45162-45174Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 20Izumikawa M. Shipley P.R. Hopke J.N. O'Hare T. Xiang L. Noel J.P. Moore B.S. J. Ind. Microbiol. Biotechnol. 2003; 30: 510-515Crossref PubMed Scopus (54) Google Scholar) and a gene of unknown function designated open reading frame 3. This three-gene operon is highly conserved and present in many Streptomyces species (18Cortes J. Velasco J. Foster G. Blackaby A.P. Rudd B.A. Wilkinson B. Mol. Microbiol. 2002; 44: 1213-1224Crossref PubMed Scopus (58) Google Scholar). Homodimeric type III polyketide synthases are involved in biosynthetic pathways in bacteria for the assembly of small aromatic metabolites (19Austin M.A. Izumikawa M. Bowman M.E. Udwary D.W. Ferrer J.-L. Moore B.S. Noel J.P. J. Biol. Chem. 2004; 279: 45162-45174Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 21Funa N. Ohnishi Y. Fujii I. Shibuya M. Ebizuka Y. Horinouchi S. Nature. 1999; 400: 897-899Crossref PubMed Scopus (239) Google Scholar, 22Moore B.S. Hopke J.N. Chem. Biochem. 2001; 2: 35-38Google Scholar). Bacterial aromatic polyketides represent a large group of biologically active natural products whose potency is often dictated by tailoring enzymes such as oxygenases, methyltransferases, and glycosyltransferases. P450s are often associated with polyketide biosynthetic gene clusters where they catalyze late-stage stereospecific and regiospecific oxidations. In the S. coelicolor operon containing CYP158A2, the 5′-gene rppA (sco1206) whose stop codon overlaps the start codon of CYP158A2 (sco1207) encodes THN synthase. This type III polyketide synthase catalyzes the sequential conversion of five molecules of malonyl-CoA to THN, which then undergoes spontaneous oxidation to flaviolin (19Austin M.A. Izumikawa M. Bowman M.E. Udwary D.W. Ferrer J.-L. Moore B.S. Noel J.P. J. Biol. Chem. 2004; 279: 45162-45174Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 20Izumikawa M. Shipley P.R. Hopke J.N. O'Hare T. Xiang L. Noel J.P. Moore B.S. J. Ind. Microbiol. Biotechnol. 2003; 30: 510-515Crossref PubMed Scopus (54) Google Scholar). In this study, we focused on the catalytic activity of CYP158A2 and demonstrated that CYP158A2 catalyzes the oxidative coupling of two or three THN-based flaviolin molecules involving C-C coupling. To explore the molecular basis of this catalytic reaction, we have determined the crystal structures of CYP158A2 in the absence and presence of the substrate flaviolin, which provides the molecular basis for P450-catalyzed oxidative coupling reactions. Expression and Purification of CYP158A2—The gene encoding S. coelicolor A3(2) CYP158A2, engineered with four histidine codons at the C terminus, was subcloned into the Escherichia coli expression vector pET17b (Novagen, Madison, WI) using the NdeI and HindIII sites (5Lamb D.C. Skaug T. Song H-L. Jackson C.J. Podust L.M. Waterman M.R. Kell D.B. Kelly D.E. Kelly S.L. J. Biol. Chem. 2002; 277: 24000-24005Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Recombinant proteins were expressed in E. coli HMS174 (DE3). Transformed E. coli was grown at 37 °C in of containing the at with and the of to a of for heme was to for at °C and the were by for at The was in of containing and The CYP158A2 were by and chromatography. The was with of and of with The was with containing and The CYP158A2 was a with of containing and and then with a of in were by and using a E. coli and were expressed in E. coli HMS174 and by as Waterman M.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were using a The of flaviolin with CYP158A2 was by of the heme CYP158A2 in was two at a was established and and sequential of a of flaviolin in in were to the to a ligand in the of of in was to the and the was The was from of were by of and were in of containing and flaviolin was from E. coli synthase as (19Austin M.A. Izumikawa M. Bowman M.E. Udwary D.W. Ferrer J.-L. Moore B.S. Noel J.P. J. Biol. Chem. 2004; 279: 45162-45174Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar) or from established A.P. J. Chem. 1991; Scopus Google Scholar, Chem. Commun. 2: Google Scholar). Following of this for on the was in a at 37 The was by the of to a of and was for in a at which the was with of and three with of The were a of the residues were in of and of was spectrometry for UV was at were as the of products from flaviolin, multiple of flaviolin with CYP158A2 were The of CYP158A2 of and and flaviolin and in a of of containing The for and then the was by and three into of The was from the in The was in and by on a with a from of in water to of in at a of with UV at The were and a of of spectrometry mass were using a mass in the with of °C and The and mass were to were using a with a from of in water to of in by with for at a of NMR were on a at a of and a of were in or and are as with to at and or at and of the were at °C using such as correlated heteronuclear multiple quantum and heteronuclear multiple bond were also used for and of CYP158A2 were using the in which of a was with of and within The used as for these at liquid was the with The to the group with as a and Full were at at the at the The x-ray were and with the W. Scopus Google Scholar). The was by molecular using the P. R.W. J. M. Biol. PubMed Scopus Google Scholar) and the CYP158A2 as a The model was in S.W. M. A. 1991; PubMed Scopus Google Scholar), and was using P. R.W. J. M. Biol. PubMed Scopus Google Scholar). were two molecules of CYP158A2 in the in a of of CYP158A2 and flaviolin were grown using a well containing and of with were within The to the group with of a and The flaviolin complex was also by molecular using the P. R.W. J. M. Biol. PubMed Scopus Google Scholar) and the CYP158A2 as a was using P. R.W. J. M. Biol. PubMed Scopus Google Scholar), and and were by Biol. 2004; PubMed Scopus Google Scholar). for crystal structures are in I. The and associated have been with the and The CYP158A2 used as a model for structures was by in the presence of that to the heme This was to and it is to the CYP158A2, the function of oxidative coupling of flaviolin is and for the resolution in for the resolution in for the resolution in for the resolution in of used in of water and residues of and residues of flaviolin in bond in bond for the resolution in and residues of and residues of flaviolin molecules in a and of that CYP158A2 flaviolin a type and that this substrate can the active site. The was suggesting the presence of one flaviolin to the of the of CYP158A2 by mass spectrometry four products from flaviolin three with a mass of and one with a mass of The molecular of and are of the dimer and trimer of flaviolin, The for flaviolin oxidation was to on the of substrate the in used activity of CYP158A2 by and reactions were as the and using CYP158A2 and flaviolin at 37 °C for major products are and as in A The structures of the flaviolin and were from multiple NMR which and COSY, HMQC, and and NMR of flaviolin were in and used as a in the characterization of and The at was to and the two at and were to and The NMR small of and were a from and also in the NMR these could by NMR of two and two at for for for and for for four suggesting that is a flaviolin C-C dimer A and of the and and established as the other was a as by NMR and which two at for and for This was as R.D. M.H. Biochem. Scopus Google Scholar), which was the of the COSY, HMQC, and of the determined by x-ray a with to many other P450 structures J.A. J. PubMed Scopus Google Scholar, M.R. M.A. C.D. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). This the substrate to the active of the A.R. A.M. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). In the of the (1.75 the are of the active that the a the F and of the absence of the substrate is with water The water the heme at the is present with a bond of of the for the of the that the substrate closes with to the The of for the upon binding of flaviolin to CYP158A2 as with the is which conformational the conformational in the and O. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar), and and and which represent of the of these and are located on the of the The most change in the and the of residues is at and where the are substrate is In the flaviolin the F the and into the active and the adjacent G is into two and a by the of this G The is present in the The residues in the into the active to the In there are in residues the and as well as in the with to the The conformational which are largely to the of the to the of the substrate and to for substrate In one change of the C on the is This the C to the which in P450s is to have important in the and in J.A. J. Full Text Full Text PDF Scopus Google Scholar). The other two conformational and are in the of result from this is that there are two molecules of flaviolin present in the active to for the of the The of the three of flaviolin are due to the high resolution of this complex (1.62 The of these two flaviolin molecules are and at PubMed Scopus Google Scholar). The of flaviolin in the of the aromatic of the flaviolin with the heme The is the from the of the heme the The of the flaviolin the and oxygen and to the heme and which in the of substrate for to in P450s J. Mol. Biol. PubMed Scopus Google Scholar, Biol. 2: PubMed Scopus Google Scholar). The and in the flaviolin are the and from the heme The binding of the flaviolin leads to of the water from the heme and the of the substrate The two flaviolin molecules are by water molecules water molecules bond with the two flaviolin for example, with and with with with and with with with and with and and are involved in with flaviolin which to the of the of substrate The water molecules are also to form of with adjacent residues and water molecules in the active site, which the substrate the of and and the of also form with the two flaviolin molecules The of and are the active of the and the group of forms with the of in the flaviolin also forms with the of and with the in the flaviolin that is a for CYP158A2 substrate The group of forms with the of and the of the is also in the flaviolin and the backbone atom of provide a environment to the substrate in the active site, also in complex M.R. S. D. C.D. 2003; PubMed Scopus Google Scholar). of flaviolin molecules also important in the water molecules in the active of the S. coelicolor A3(2) is a model actinomycete that produces >20 secondary metabolites including antibiotics, siderophores, and other (4Bentey S.D. Chater K.F. Cerdeno-Tarraga A.M. Challis G.L. Thomson N.R. James K.D. Harris D.E. Quail M.A. Kieser H. Harper D. Bateman A. Brown S. Chandra G. Chen C.W. Collins M. Cronin A. Fraser A. Goble A. Hidalgo J. Hornsby T. Howarth S. Huang C.H. Kieser T. Larke L. Murphy L. Oliver K. O'Neil S. Rabbiowitsch E. Rajandream M.A. Rutherford K. Rutter S. Seeger K. Saunders D. Sharp S. Squares R. Squares S. Taylor K. Warren T. Wietzorrek A. Woodward J. Barrell B.G. Parkhill J. Hopwood D.A. Nature. 2002; 417: 141-147Crossref PubMed Scopus (2600) Google Scholar). A of these compounds are in by the function of P450 the functions of the 18 S. coelicolor are at all from genome is predicted to molecules from the environment on of from Streptomyces M. D.C. R. M. Kelly S.L. Biochem. Commun. 1999; PubMed Scopus (58) Google Scholar). can participate in biosynthesis in L.M. Y. M. B.A. H. D.C. Kelly S.L. Waterman M.R. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), such are by S. coelicolor A3(2). on in other Streptomyces on the biosynthesis of THN-based red-brown pigments (18Cortes J. Velasco J. Foster G. Blackaby A.P. Rudd B.A. Wilkinson B. Mol. Microbiol. 2002; 44: 1213-1224Crossref PubMed Scopus (58) Google Scholar, 21Funa N. Ohnishi Y. Fujii I. Shibuya M. Ebizuka Y. Horinouchi S. Nature. 1999; 400: 897-899Crossref PubMed Scopus (239) Google Scholar), we flaviolin as a substrate for CYP158A2 and established the endogenous function of a S. coelicolor A3(2) CYP in secondary have we shown a function of CYP158A2 to and flaviolin in we have found that two flaviolin molecules the active in the In the CYP158A2 with two flaviolin the of and in the active are To two flaviolin the of is the heme to that is also to the oxygen of and that with and which a bond to with and the oxygen of This leads to the of the of the the active site, which to water The flaviolin-bound conformational with to the form that the and the of the and in two flaviolin molecules with and and the and in flaviolin molecules to important in the of the water in the substrate binding of the on these the of the for to the and the may for as has been suggested for K. K. H. Chem. PubMed Scopus Google Scholar), or of water molecules for of The absence of one or of these two hydroxyl may or the which for the The of of water molecules in the binding of hydroxyl and of flaviolin the of such in the binding of substrates and in the conformational they have In the crystal that CYP158A2 may also have the to a substrate CYP158A2 has a large active and there is a large of the of the four products is a flaviolin This result that flaviolin dimer and flaviolin can to the active at the which is the for trimer The P450 catalytic P.R. Cytochrome and Google Scholar) is a and can as substrate and of the bond with of a complex of with substrate to form and The of of the flaviolin has been established at this of coupling by P450s have been R. Zenk M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the coupling of has been as a The flaviolin atom to the P450 atom in the crystal is the products we are and A can that oxidation of the flaviolin by atom the in the crystal The is to from the which is located from the in the The atom is located the of a or a there with leads to the of the or at and at or The shown has at and also and which may also that the multiple products were the can with at the atom F.P. Chem. 2001; PubMed Scopus Google Scholar), which a a at In this this with the bond of the adjacent flaviolin A of the in is that the flaviolin with the flaviolin in the active that form or within the active site. could the P450 and to form in of the The products the and of of the P450 active site. this we of the In we the endogenous substrate identified in the P450s from S. CYP158A2 can flaviolin to red-brown which may to the the of UV to which this soil bacterium is (18Cortes J. Velasco J. Foster G. Blackaby A.P. Rudd B.A. Wilkinson B. Mol. Microbiol. 2002; 44: 1213-1224Crossref PubMed Scopus (58) Google Scholar). In combination with the structures provide into the of the oxidative coupling reactions of in for of highly and I. and Hopwood for and the at the at for M. and for with mass H. and for on for x-ray and for form with