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Crystal Structure of a Bacterial Type III Polyketide Synthase and Enzymatic Control of Reactive Polyketide Intermediates

2004; Elsevier BV; Volume: 279; Issue: 43 Linguagem: Inglês

10.1074/jbc.m406567200

ISSN

1083-351X

Autores

M.B. Austin, Miho Izumikawa, M.E. Bowman, Daniel W. Udwary, Jean‐Luc Ferrer, Bradley S. Moore, Joseph P. Noel,

Tópico(s)

Plant-Microbe Interactions and Immunity

Resumo

In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthlene synthase (THNS) catalyzes the iterative condensation of five CoA-linked malonyl units to form a pentaketide intermediate. THNS subsequently catalyzes dual intramolecular Claisen and aldol condensations of this linear intermediate to produce the fused ring tetrahydroxynaphthalene (THN) skeleton. The type III PKS-catalyzed polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic triad in an internal active site cavity, is fairly well understood. However, the mechanistic basis for the unusual production of THN and dual cyclization of its malonyl-primed pentaketide is obscure. Here we present the first bacterial type III PKS crystal structure, that of Streptomyces coelicolor THNS, and identify by mutagenesis, structural modeling, and chemical analysis the unexpected catalytic participation of an additional THNS-conserved cysteine residue in facilitating malonyl-primed polyketide extension beyond the triketide stage. The resulting new mechanistic model, involving the use of additional cysteines to alter and steer polyketide reactivity, may generally apply to other PKS reaction mechanisms, including those catalyzed by iterative type I and II PKS enzymes. Our crystal structure also reveals an unanticipated novel cavity extending into the "floor" of the traditional active site cavity, providing the first plausible structural and mechanistic explanation for yet another unusual THNS catalytic activity: its previously inexplicable extra polyketide extension step when primed with a long acyl starter. This tunnel allows for selective expansion of available active site cavity volume by sequestration of aliphatic starter-derived polyketide tails, and further suggests another distinct protection mechanism involving maintenance of a linear polyketide conformation. In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthlene synthase (THNS) catalyzes the iterative condensation of five CoA-linked malonyl units to form a pentaketide intermediate. THNS subsequently catalyzes dual intramolecular Claisen and aldol condensations of this linear intermediate to produce the fused ring tetrahydroxynaphthalene (THN) skeleton. The type III PKS-catalyzed polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic triad in an internal active site cavity, is fairly well understood. However, the mechanistic basis for the unusual production of THN and dual cyclization of its malonyl-primed pentaketide is obscure. Here we present the first bacterial type III PKS crystal structure, that of Streptomyces coelicolor THNS, and identify by mutagenesis, structural modeling, and chemical analysis the unexpected catalytic participation of an additional THNS-conserved cysteine residue in facilitating malonyl-primed polyketide extension beyond the triketide stage. The resulting new mechanistic model, involving the use of additional cysteines to alter and steer polyketide reactivity, may generally apply to other PKS reaction mechanisms, including those catalyzed by iterative type I and II PKS enzymes. Our crystal structure also reveals an unanticipated novel cavity extending into the "floor" of the traditional active site cavity, providing the first plausible structural and mechanistic explanation for yet another unusual THNS catalytic activity: its previously inexplicable extra polyketide extension step when primed with a long acyl starter. This tunnel allows for selective expansion of available active site cavity volume by sequestration of aliphatic starter-derived polyketide tails, and further suggests another distinct protection mechanism involving maintenance of a linear polyketide conformation. 1,3,6,8-Tetrahydroxynaphthalene (THN) 1The abbreviations used are: THN, 1,3,6,8-tetrahydroxynaphthalene; PKS, polyketide synthase; THNS, THN synthase; CHS, chalcone synthase; STS, stilbene synthase; TAL, triacetic acid lactone (4-hydroxy-6-methyl- 2H-pyran-2-one); 2-PS, 2-pyrone (TAL) synthase; PEG, polyethylene glycol; KS or KAS, β-ketoacyl synthase; MtFabH, an M. tuberculosis KAS III enzyme; MtPKS18, M. tuberculosis PKS18; HPLC, high performance liquid chromatography; MOPSO, (3-N-morpholino)-2-hydroxypropanesulfonic acid). is biosynthesized from polyketide intermediates in fungi and bacteria (Fig. 1A). In some fungi, THN is reduced to 1,8-dihydroxynaphthalene and undergoes polymerization to form UV-protective 1,8-dihydroxynaphthalene-melanin (1Feng B. Wang X. Hauser M. Kaufmann S. Jentsch S. Haase G. Becker J.M. Szaniszlo P.J. Infect. Immun. 2001; 69: 1781-1794Crossref PubMed Scopus (111) Google Scholar). Filamentous bacteria of the genus Streptomyces utilize THN not only for melanin production (2Ueda K. Kim K.M. Beppu T. Horinouchi S. J. Antibiot. (Tokyo). 1995; 48: 638-646Crossref PubMed Scopus (36) Google Scholar) but also incorporate the THN scaffold into pharmacologically active meroterpenoids such as neomarinone (3Kalaitzis J.A. Hamano Y. Nilsen G. Moore B.S. Org. Lett. 2003; 5: 4449-4452Crossref PubMed Scopus (51) Google Scholar). Fungi and bacteria use dramatically different enzymatic systems to produce THN from five activated thioester-linked malonyl building blocks. In fungi, THN is synthesized by an iterative type I polyketide synthase (PKS), which functions as a large (∼230-kDa) multidomain complex (4Fujii I. Mori Y. Watanabe A. Kubo Y. Tsuji G. Ebizuka Y. Biochemistry. 2000; 39: 8853-8858Crossref PubMed Scopus (87) Google Scholar). Whereas type I PKSs and their fatty acid synthase ancestors have been intensely studied, their three-dimensional domain organization remains obscure due to their size and complexity. In stark contrast, bacteria use a structurally simple type III PKS architecturally organized as a homodimeric condensing enzyme (∼40 kDa) to synthesize THN from five coenzyme A (CoA)-tethered malonyl units, without the use of additional enzymes or catalytic domains (5Funa N. Ohnishi Y. Fujii I. Shibuya M. Ebizuka Y. Horinouchi S. Nature. 1999; 400: 897-899Crossref PubMed Scopus (239) Google Scholar, 6Cortes J. Velasco J. Foster G. Blackaby A.P. Rudd B.A.M. Wilkinson B. Mol. Microbiol. 2002; 44: 1213-1224Crossref PubMed Scopus (58) Google Scholar, 7Funa N. Ohnishi Y. Ebizuka Y. Horinouchi S. Biochem. J. 2002; 367: 781-789Crossref PubMed Google Scholar, 8Izumikawa 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). The type III PKS enzyme family (9Schröder J. Trends Plant Sci. 1997; 2: 373-378Abstract Full Text PDF Google Scholar, 10Schröder J. Recent Adv. Phytochem. 2000; 34: 55-89Crossref Scopus (20) Google Scholar, 11Austin M.B. Noel J.P. Nat. Prod. Rep. 2003; 20: 79-110Crossref PubMed Scopus (736) Google Scholar), defined by homology to chalcone synthase (CHS) (12Kreuzaler F. Hahlbrock K. FEBS Lett. 1972; 28: 69-72Crossref PubMed Scopus (95) Google Scholar, 13Kreuzaler F. Hahlbrock K. Arch. Biochem. Biophys. 1975; 169: 84-90Crossref PubMed Scopus (64) Google Scholar, 14Heller W. Hahlbrock K. Arch. Biochem. Biophys. 1980; 200: 617-619Crossref PubMed Scopus (129) Google Scholar, 15Reimold U. Kroeger M. Dreuzaler F. Hahlbrock K. EMBO J. 1983; 2: 1801-1805Crossref PubMed Google Scholar), is currently known to include at least 15 functionally divergent (11Austin M.B. Noel J.P. Nat. Prod. Rep. 2003; 20: 79-110Crossref PubMed Scopus (736) Google Scholar) β-ketosynthases (KSs) of plant (9Schröder J. Trends Plant Sci. 1997; 2: 373-378Abstract Full Text PDF Google Scholar, 10Schröder J. Recent Adv. Phytochem. 2000; 34: 55-89Crossref Scopus (20) Google Scholar, 11Austin M.B. Noel J.P. Nat. Prod. Rep. 2003; 20: 79-110Crossref PubMed Scopus (736) Google Scholar) and bacterial origin (11Austin M.B. Noel J.P. Nat. Prod. Rep. 2003; 20: 79-110Crossref PubMed Scopus (736) Google Scholar, 16Moore B.S. Hopke J.N. ChemBioChem. 2001; 2: 35-38Crossref PubMed Scopus (99) Google Scholar). As first revealed in the crystal structure of alfalfa CHS (17Ferrer J.L. Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Nat. Struct. Biol. 1999; 6: 775-784Crossref PubMed Scopus (573) Google Scholar), each type III PKS monomer utilizes a Cys-His-Asn catalytic triad (Fig. 1B) within an internal active site cavity that is connected to the surrounding aqueous phase by a narrow CoA-binding tunnel. The catalytic triad and the buried active site cavity condense an acetyl unit (derived from the decarboxylation of a malonyl-CoA) to a preloaded starter molecule attached by a thioester bond to the catalytic cysteine (18Lanz T. Tropf S. Marner F.J. Schröder J. Schröder G. J. Biol. Chem. 1991; 266: 9971-9976Abstract Full Text PDF PubMed Google Scholar). Subsequent structure-guided mutagenic and biochemical studies of CHS provided a clear picture of the conserved type III PKS polyketide extension mechanism (Fig. 1B) (19Jez J.M. Ferrer J.L. Bowman M.E. Dixon R.A. Noel J.P. Biochemistry. 2000; 39: 890-902Crossref PubMed Scopus (284) Google Scholar, 20Jez J.M. Noel J.P. J. Biol. Chem. 2000; 275: 39640-39646Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 21Suh D.Y. Kagami J. Fukuma K. Sankawa U. Biochem. Biophys. Res. Commun. 2000; 275: 725-730Crossref PubMed Scopus (46) Google Scholar). Type III PKSs are both iterative and multifunctional, having evolved to catalyze an impressive repertoire of functionally divergent and mechanistically complex reactions. These remarkable enzymes typically perform three polyketide extensions of their preferred CoA-activated starter molecules (ranging in size from acetyl- to caffeoyl-CoA), prior to catalyzing six-membered ring formation via intramolecular Claisen-, aldol-, or lactone-forming cyclization reactions (11Austin M.B. Noel J.P. Nat. Prod. Rep. 2003; 20: 79-110Crossref PubMed Scopus (736) Google Scholar). Mutagenic analyses of plant PKSs based upon homology with CHS or upon the more recent crystal structures of daisy 2-pyrone synthase (2-PS) (22Jez J.M. Austin M.B. Ferrer J. Bowman M.E. Schröder J. Noel J.P. Chem. Biol. 2000; 7: 919-930Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) and pine stilbene synthase (STS) (23Austin M.B. Bowman M.E. Ferrer J. Schröder J. Noel J.P. Chem. Biol. 2004; (in press)PubMed Google Scholar), facilitated identification of much of the structural and mechanistic underpinnings for plant type III PKS substrate specificity and catalysis (11Austin M.B. Noel J.P. Nat. Prod. Rep. 2003; 20: 79-110Crossref PubMed Scopus (736) Google Scholar). Despite these advances, the structural and mechanistic basis for the bacterial THNS reaction pathway remains frustratingly obscure. Whereas most type III PKSs cyclize linear tetraketide intermediates to form phloroglucinol- or resorcinol-based products, THNS uniquely catalyzes two successive intramolecular carbon-carbon cyclization reactions of an all malonate-derived pentaketide chain (Fig. 1A). How THNS controls chain length and reactivity while avoiding the many possible derailment reactions available to its carboxylated polyketide intermediate is one of this enzyme's most intriguing features. Furthermore, it is unclear which of several possible cyclization routes to THN are utilized by the enzyme (Fig. 1C), despite the perpetuation in the literature of a hypothetical STS-like initial C-2 → C-7 intramolecular aldol condensation (5Funa N. Ohnishi Y. Fujii I. Shibuya M. Ebizuka Y. Horinouchi S. Nature. 1999; 400: 897-899Crossref PubMed Scopus (239) Google Scholar). Our recent structural and mechanistic analysis of STS (23Austin M.B. Bowman M.E. Ferrer J. Schröder J. Noel J.P. Chem. Biol. 2004; (in press)PubMed Google Scholar), considered in light of earlier biomimetic studies (24Harris T.M. Harris C.M. Pure Appl. Chem. 1986; 58: 283-294Crossref Scopus (46) Google Scholar), instead implicates the C-1 thioester carbonyl and the C-10 methylene (activated by the C-11 carboxylate moiety) as the most likely electrophile and nucleophile, respectively, for intramolecular cyclization. Thus, an initial CHS-like C-6 → C-1 Claisen condensation could lead to a prearomatic phloroglucinol-like intermediate, or a C-10 → C-5 aldol condensation could lead to the corresponding resorcinol-like intermediate (Fig. 1C). It is also possible that the THNS active site may juxtapose these two reactive carbons, C-1 and C-10, to first facilitate an unprecedented C-10 → C-1 Claisen condensation. Although the resulting 10-membered polyketide ring seems energetically unfavorable relative to a more stable six-membered ring, in the context of the THNS active site, it could form and rapidly collapse via any one of five symmetric intramolecular aldol condensations to form the two stable fused six-carbon rings of the THN product (Fig. 1C). Significantly, no on-pathway monocyclic intermediates or their stable aromatic derivatives have ever been observed to result from the THNS-catalyzed reaction, suggesting that the unknown initial cyclic intermediate must indeed strongly facilitate enzymatic formation of the second ring. Although homology-based analysis of bacterial THNSs predicts a number of unusual active site residues (11Austin M.B. Noel J.P. Nat. Prod. Rep. 2003; 20: 79-110Crossref PubMed Scopus (736) Google Scholar, 25Moore B.S. Hertweck C. Hopke J.N. Izumikawa M. Kalaitzis J.A. Nilsen G. O'Hare T. Piel J. Shipley P.R. Xiang L. Austin M.B. Noel J.P. J. Nat. Prod. 2002; 65: 1956-1962Crossref PubMed Scopus (87) Google Scholar), including four or five exposed cysteine side chain thiol groups that might contribute to these enzymes' unusual physiological reaction, sequence divergence between bacterial CHS-like enzymes and the structurally characterized plant PKSs increases the likelihood of significant model bias and error in homology models derived from distantly related plant PKS structures. In order to illuminate the mechanistic features responsible for these enzymes' catalytic pathway, we undertook a structural and biochemical analysis of THNS from the model actinomycete Streptomyces coelicolor A3 (2Ueda K. Kim K.M. Beppu T. Horinouchi S. J. Antibiot. (Tokyo). 1995; 48: 638-646Crossref PubMed Scopus (36) Google Scholar, 26Bentley 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. Rabbinowitsch 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 (2602) Google Scholar). Toward this end, we present here the 2.2-Å crystal structure of THNS from S. coelicolor A3 (2Ueda K. Kim K.M. Beppu T. Horinouchi S. J. Antibiot. (Tokyo). 1995; 48: 638-646Crossref PubMed Scopus (36) Google Scholar), accompanied by additional mutagenic and biochemical investigations into the complicated THNS reaction mechanism. Expression, Purification, and Mutagenesis—The construction and characterization of the S. coelicolor THNS construct in the pHIS8 vector was previously reported (8Izumikawa 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). Mutations were introduced using mutagenic oligonucleotides (Table I) and the QuikChange (Stratagene) system. The entire coding sequences of mutant constructs were verified by nucleotide sequencing. Wild type and mutant proteins were expressed and purified as previously described (8Izumikawa 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). For crystallization, THNS was overexpressed in Escherichia coli BL21(DE3) cells containing the pLysS plasmid (Invitrogen), purified, buffer-exchanged, and concentrated as described for CHS (19Jez J.M. Ferrer J.L. Bowman M.E. Dixon R.A. Noel J.P. Biochemistry. 2000; 39: 890-902Crossref PubMed Scopus (284) Google Scholar), resulting in final enzyme concentrations of 5–50 mg/ml in 12 mm HEPES (pH 7.5), 25 mm NaCl, and 5 mm dithiothreitol. Aliquots were stored at -80 °C.Table IMutagenesis of THNS active site cysteines The mutated codon is underlined.MutantMutagenic oligonucleotide sequenceC106S5′-G ATC ATC TAC GTC TCC TCC ACG GGC TTC ATG ATG CC-3′C168S5′-C GTG GCC TGC GAG TTC TCC TCG CTG TGC TAC CAG CC-3′C171S5′-GC GAG TTC TGC TCG CTG TCC TAC CAG CCC ACC GAC CTC G-3′C184S5′-GC GTG GGC TCC CTG CTC TCC AAC GGC CTC TTC GGC G-3′ Open table in a new tab Crystallization and Data Collection—THNS was crystallized by vapor diffusion in hanging drops consisting of a 1:1 mixture of purified protein solution and crystallization buffer. The crystallization buffer contained 14% (w/v) PEG 8000, 200 mm MgCl2, 100 mm Na+-MOPSO buffer (pH 7.0), 5 mm dithiothreitol, and 3% (w/v) sucrose. A 30-s crystal soak prior to freezing employed a cryogenic solution differing from the crystallization buffer due to an increased PEG 8000 concentration (16% (w/v)) and the inclusion of 20% (v/v) glycerol. For heavy atom derivatization, native THNS crystals were soaked overnight in crystallization buffer differing from the crystallization solution due to an increased PEG 8000 concentration (16% (w/v)) and the inclusion of 0.1 mm K2PtCl4 prior to cryogenic freezing as before. Several unsuccessful heavy atom soaks were screened for anomalous diffraction at the European Synchrotron Radiation Facility, whereas the 2.2-Å native and 2.9-Å K2PtCl4 derivative data sets were collected at the Stanford Synchrotron Radiation Laboratory. Images were indexed and integrated with DENZO (27Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar), reflections were merged with SCALEPACK (27Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar), and data reduction was completed with CCP4 programs (28Dodson E.J. Winn M. Ralph A. Methods Enzymol. 1997; 277: 620-633Crossref PubMed Scopus (176) Google Scholar). THNS crystallizes in the P2(1) space group, with unit cell dimensions of a = 76.68 Å, b = 69.68 Å, c = 81.14 Å, α = γ = 90°, and β = 95.42°. Structure Determination and Refinement—The S. coelicolor THNS structure was solved from the 2.9-Å platinum derivative data set. Experimental multiple wavelength anomalous dispersion phases were obtained using SOLVE (29Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar), based upon two K2PtCl4 sites. The experimental electron density maps were improved by bulk solvent density modification and automated building with RESOLVE (29Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). Inspection of electron density maps and subsequent model building were performed in O (30Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 148-157Crossref PubMed Scopus (2) Google Scholar). The asymmetric unit contains one physiological THNS dimer. Two copies of a monomeric THNS homology model, generated by MODELLER (31Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10565) Google Scholar) from an alignment with the alfalfa CHS2 crystal structure (17Ferrer J.L. Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Nat. Struct. Biol. 1999; 6: 775-784Crossref PubMed Scopus (573) Google Scholar), were manually inserted into the density-modified electron density maps and rebuilt in O. Following the success of a single round of refinement in CNS (32Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice R.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar) of this rebuilt protein model against the same 2.9-Å platinum derivative data set, subsequent iterative rounds of refinement in CNS utilized the higher quality 2.2-Å native data set (Table II). Coordinate and parameter files for the PEG and glycerol ligands were obtained from the HIC-Up (Hetero-compound Information Centre-Uppsala) site on the World Wide Web (x-ray.bmc.uu.se/hicup/). PROCHECK (in CCP4) analysis of the final refined crystal structure (see Table II) revealed 86.6% of residue conformations to be in the most favored region of the Ramachandran plot, with 11.2% in additional allowed, 2.2% in generously allowed, and no residues in disallowed regions. THNS residues in generously allowed conformations are located in poorly ordered surface loops. Coordinates and structure factors for THNS have been deposited in the Protein Data Bank (1U0M). Structures were overlaid for comparison using MIDAS (33Ferrin T.E. Huang C.C. Jarvis L.E. Langridge R. J. Mol. Graph. 1988; 6: 13-27Crossref Scopus (929) Google Scholar). Structural illustrations were prepared with MOLSCRIPT (34Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and rendered with POV-Ray (persistence of vision ray tracer; available on the World Wide Web at www.povray.org).Table IICrystallographic data and refinement statistics for the S. coelicolor THNS structureNativePlatinum derivativeλ1λ2λ3Wavelength (Å)0.7731.07181.07130.9050Resolution (Å)2.22.95Space groupP2(1)P2(1)Unit cell dimensions (Å)a = 76.7a = 76.5b = 69.7b = 69.9c = 81.1c = 81.2Unit cell dimensions (degrees)β = 95.4β = 95.3Total reflections139,86992,789102,36087,984Unique reflections37,66632,80235,27031,799CompletenessaThe number in parenthesis is for the highest resolution shell (%)89.8 (20.6)87.8 (52.0)93.9 (76.2)84.9 (46.7)I/σaThe number in parenthesis is for the highest resolution shell25.5 (4.9)15.0 (2.3)13.1 (1.8)13.9 (1.8)RsymaThe number in parenthesis is for the highest resolution shell,bRsym = Σ|Ih - 〈Ih〉|/ΣIh, where 〈Ih〉 is the average intensity over symmetry equivalent reflections5.8 (11.9)9.9 (37.9)10.4 (50.7)10.6 (49.1)Heavy atom sites2Phasing figure of merit0.49Refinement statisticsRcrystcR-factors (Rcryst) = Σ|Fo - Fc|/ΣFo, where summation is over the data used for refinement/RfreedRfree, same definition as for Rcryst, but it includes only 5% of data excluded from refinement (%)25.2/29.3Protein atoms5308Ligand atoms50Water molecules148Root mean square deviation bond lengths (Å)0.008Root mean square deviation bond angles (degrees)1.4Average B-factor, protein (Å2)54.0Average B-factor, solvent (Å2)67.0a The number in parenthesis is for the highest resolution shellb Rsym = Σ|Ih - 〈Ih〉|/ΣIh, where 〈Ih〉 is the average intensity over symmetry equivalent reflectionsc R-factors (Rcryst) = Σ|Fo - Fc|/ΣFo, where summation is over the data used for refinementd Rfree, same definition as for Rcryst, but it includes only 5% of data excluded from refinement Open table in a new tab Enzyme Activities—THNS activity was monitored by reversed-phase HPLC (8Izumikawa 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 TLC. Reversed-phase TLC analysis of flaviolin/TAL product ratios were obtained from standardized 50-μl room temperature reactions containing 100 mm Tris-HCl buffer (pH 7.5), 10 μm THNS, and 20 μm [2-14C]malonyl-CoA (PerkinElmer Life Sciences). Reactions were quenched with 5 μl of concentrated HCl and extracted twice with 50 μl of ethyl acetate. The extracts were dried, dissolved in 10 μl of MeOH, applied to Merck C18-silica TLC plates, developed in MeOH/H2O/AcOH (60:40:1, v/v/v), and visualized with radiography film. Apparent kinetic constants kcat and Km for THN production (Table III) were determined from Eadie-Hofstee plots, using an average of three or more independent assays (8Izumikawa 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).Table IIISteady state kinetic constants for THN production for wild type THNS and the Cys to Ser mutantsWild typeC106SC168SC171SC184Skcat × 102 (min-1)27.4 ± 2.4NAaNA, no activityNDbND, not determined27.9 ± 1.347.5 ± 5.4Km (μm)2.3 ± 0.6NAND8.5 ± 1.01.4 ± 0.5kcat/Km (s-1m-1)1,970NAND5495,530a NA, no activityb ND, not determined Open table in a new tab Flaviolin Labeling and 13C NMR Analysis—E. coli BL21(DE3)pLysS cells (Invitrogen) harboring the pHIS8-THNS construct were grown at 37 °C in 1.2 liters of Terrific broth containing 50 μg/ml kanamycin and 37 μg/ml chloramphenicol until A600 reached 0.7. Following induction with 0.5 mm isopropyl β-d-1-thiogalactopyranoside and the addition of 120 mg of [1,2-13C]AcONa (Cambridge Isotope Laboratories, Inc.), cultures were shaken at 28 °C overnight, with additional 120-mg aliquots of [1,2-13C]AcONa added 3 and 6 h after induction for a total of 360 mg of labeled acetate. Cells were removed by centrifugation. The media supernatant was acidified with concentrated HCl and extracted with ethyl acetate. The crude organic extract was concentrated and separated by oxalic acid-treated silica flash chromatography employing a stepwise chloroform to methanol gradient. The 90% methanol fraction was further purified by preparative HPLC (YMC ODS-AQ 10 × 250-mm reversed-phase column with a linear solvent gradient of 0.15% trifluoroacetic acid in water to methanol over 60 min; flow rate = 2.0 ml/min). 2.5 mg of enriched flaviolin in Me2SO-d6 was analyzed by 13C INADEQUATE NMR on a Bruker 600-MHz spectrometer. THNS Crystal Structure—We were unable to solve the THNS crystal structure by molecular replacement using a 2.2-Å native data set and homology models based on plant type III PKS structures. Moreover, the in vivo incorporation of selenomethionine into the E. coli-expressed S. coelicolor THNS did not result in stable and active protein preparations. Fortunately, a 2.9-Å data set of a K2PtCl4 soak of our native crystals produced an anomalous signal suitable for phasing the structure using multiple anomalous diffraction with SOLVE (29Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). Two platinum sites producing strong peaks on anomalous difference Patterson maps permitted phasing as well as phase extension and phase improvement by density modification using RESOLVE (29Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3220) Google Scholar). The quality of the phase information deteriorated significantly beyond 3.9 Å, but the initial figure of merit-weighted 2Fo - Fc electron density map was interpretable. Whereas RESOLVE automatic model building using this low resolution phasing information produced only a few erroneous polyalanine chains, their symmetrical distribution in the asymmetric unit facilitated identification of electron density corresponding to the THNS conserved type III PKS αβαβα-fold core domain and expected physiological homodimeric interface (11Austin M.B. Noel J.P. Nat. Prod. Rep. 2003; 20: 79-110Crossref PubMed Scopus (736) Google Scholar). A CHS-derived THNS homology model was then globally positioned in this experimental electron density map, and each residue with interpretable electron density was individually repositioned using O. Following a single round of successful CNS refinement of our manually placed and partially rebuilt model against the 2.9-Å derivative data, the resulting 2Fo - Fc and Fo - Fc electron density maps indicated a number of regions needing to be rebuilt. In order to move rapidly and achieve a higher quality final model, subsequent rounds of refinement utilized our original native data set extending to 2.2-Å resolution. Portions of the model with no apparent electron density (discussed below) were initially deleted, but most were eventually replaced, since later electron density maps greatly improved following iterative rounds of building and refinement using the 2.2-Å native data set. Although previous plant PKS-based homology modeling failed to predict the many subtle backbone differences observed throughout this first bacterial type III PKS crystal structure reported here, there are no drastic rearrangements of the conserved αβαβα-fold or dimer interface in THNS. Our crystal structure shows that homology-based assignments of the THNS catalytic triad and other active site residues are essentially accurate, as previously supported by in vitro assays of Streptomyces griseus THNS point mutants (7Funa N. Ohnishi Y. Ebizuka Y. Horinouchi S. Biochem. J. 2002; 367: 781-789Crossref PubMed Google Scholar). Interestingly, THNSs feature unusual (∼25-residue) extensions of their C termini, not found in plant or most other bacterial CHS-like sequences examined to date. These additional residues may facilitate in vivo protein-protein interactions with upstream and downstream enzymes, including the P450 or ORF3 proteins encoded next to the THNS gene in several gene clusters obtained from Streptomyces species (2Ueda K. Kim K.M. Beppu T. Horinouchi S. J. Antibiot. (Tokyo). 1995; 48: 638-646Crossref PubMed Scopus (36) Google Scholar, 6Cortes J. Velasco J. Foster G. Blackaby A.P. Rudd B.A.M. Wilkinson B. Mol. Microbiol. 2002; 44: 1213-1224Crossref PubMed Scopus (58) Google Scholar). The elimination of this C-terminal extension was previously shown to have no detrimental effect

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