Title: Structure and Functional Analysis of RifR, the Type II Thioesterase from the Rifamycin Biosynthetic Pathway
Abstract: Two thioesterases are commonly found in natural product biosynthetic clusters, a type I thioesterase that is responsible for removing the final product from the biosynthetic complex and a type II thioesterase that is believed to perform housekeeping functions such as removing aberrant units from carrier domains. We present the crystal structure and the kinetic analysis of RifR, a type II thioesterase from the hybrid nonribosomal peptide synthetases/polyketide synthase rifamycin biosynthetic cluster of Amycolatopsis mediterranei. Steady-state kinetics show that RifR has a preference for the hydrolysis of acyl units from the phosphopantetheinyl arm of the acyl carrier domain over the hydrolysis of acyl units from the phosphopantetheinyl arm of acyl-CoAs as well as a modest preference for the decarboxylated substrate mimics acetyl-CoA and propionyl-CoA over malonyl-CoA and methylmalonyl-CoA. Multiple RifR conformations and structural similarities to other thioesterases suggest that movement of a helical lid controls access of substrates to the active site of RifR. Two thioesterases are commonly found in natural product biosynthetic clusters, a type I thioesterase that is responsible for removing the final product from the biosynthetic complex and a type II thioesterase that is believed to perform housekeeping functions such as removing aberrant units from carrier domains. We present the crystal structure and the kinetic analysis of RifR, a type II thioesterase from the hybrid nonribosomal peptide synthetases/polyketide synthase rifamycin biosynthetic cluster of Amycolatopsis mediterranei. Steady-state kinetics show that RifR has a preference for the hydrolysis of acyl units from the phosphopantetheinyl arm of the acyl carrier domain over the hydrolysis of acyl units from the phosphopantetheinyl arm of acyl-CoAs as well as a modest preference for the decarboxylated substrate mimics acetyl-CoA and propionyl-CoA over malonyl-CoA and methylmalonyl-CoA. Multiple RifR conformations and structural similarities to other thioesterases suggest that movement of a helical lid controls access of substrates to the active site of RifR. Assembly line complexes, which include modular polyketide synthases (PKS) 3The abbreviations used are: PKS, polyketide synthase; NRPS, nonribosomal peptide synthetase; TEI, type I thioesterase; TEII, type II thioesterase; Ppant, phosphopantetheinyl arm; FAS, fatty acid synthase; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; SeMet, selenomethionyl; HPLC, high pressure liquid chromatography; ACP, acyl carrier protein. and nonribosomal peptide synthetases (NRPS), are multifunctional proteins composed of modules that work in succession to synthesize secondary metabolites, many of which are precursors of potent antibiotics, immunosuppressants, anti-tumor agents, and other bioactive compounds. Rifamycin, the precursor to the anti-tuberculosis drug rifampicin, is produced by the rifamycin assembly line complex, which is an NRPS/PKS hybrid system composed of one NRPS-like and 10 PKS modules (1Yu T.-W. Shen Y. Doi-Katayama Y. Tang L. Park C. Moore B.S. Richard Hutchinson C. Floss H.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9051-9056Crossref PubMed Scopus (133) Google Scholar). Each module in an assembly line complex extends and modifies the intermediate compound before passing it on to the next module in the series (Fig. 1A). The intermediate compounds are covalently attached through a thioester linkage to the phosphopantetheine arm (Ppant) of carrier domains, one associated with each module, until they are released from the synthase, usually by a type I thioesterase (TEI) (2Weissman K.J. Philos. Transact. A Math Phys. Eng. Sci. 2004; 362: 2671-2690Crossref PubMed Scopus (45) Google Scholar, 3Hutchinson C.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3010-3012Crossref PubMed Scopus (42) Google Scholar). TEIs are usually integrated into the final module of the assembly line complex and remove the final product through macrocyclization or hydrolysis. Occasionally, tandem type I thioesterases are integrated at the C terminus of the final module of NRPS pathways (4Roongsawang N. Washio K. Morikawa M. ChemBioChem. 2007; 8: 501-512Crossref PubMed Scopus (31) Google Scholar). Although TEIs are covalently attached to the terminal module and generally process only the final product of an assembly line complex, type II thioesterases (TEIIs) are discrete proteins that can remove intermediates from any module in the complex. A variety of functions have been attributed to TEIIs, the most prevalent of which is a "housekeeping function," the removal of aberrant acyl units from carrier domains. These aberrant acyl units may be due to premature decarboxylation by a PKS ketosynthase domain (5Heathcote M.L. Staunton J. Leadlay P.F. Chem. Biol. 2001; 8: 207-220Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) (Fig. 1B) or to mispriming of the carrier domain by a promiscuous phosphopantetheinyl transferase (6Schwarzer D. Mootz H.D. Linne U. Marahiel M.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14083-14088Crossref PubMed Scopus (165) Google Scholar, 7Yeh E. Kohli R.M. Bruner S.D. Walsh C.T. ChemBioChem. 2004; 5: 1290-1293Crossref PubMed Scopus (86) Google Scholar, 8Linne U. Schwarzer D. Schroeder G.N. Marahiel M.A. Eur. J. Biochem. 2004; 271: 1536-1545Crossref PubMed Scopus (31) Google Scholar) (Fig. 1C). Other proposed functions for TEIIs include the removal of intermediates from the synthase as in the case of the mammary gland rat fatty acid synthase (FAS) TEII in lactating rats, which removes medium chain C8-C12 fatty acids from the ACP domain (9Libertini L.J. Smith S. J. Biol. Chem. 1978; 253: 1393-1401Abstract Full Text PDF PubMed Google Scholar) and the removal of amino acid derivatives from a carrier domain (10Chen H. Hubbard B.K. O'Connor S.E. Walsh C.T. Chem. Biol. 2002; 9: 103-112Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 11Chen H. Walsh C.T. Chem. 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Microbiology. 2002; 148: 1777-1783Crossref PubMed Scopus (29) Google Scholar). Two models have been proposed for the TEII housekeeping function (5Heathcote M.L. Staunton J. Leadlay P.F. Chem. Biol. 2001; 8: 207-220Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In the high specificity model, the TEII scans the complex and efficiently removes only aberrant acyl units. In the low specificity model, the TEII removes both correct and incorrect acyl units from the Ppant arm at an inefficient rate. Correct acyl units are quickly incorporated into the growing intermediate compound. In contrast, incorrect acyl units stall the assembly line, providing a longer window of opportunity for removal by a TEII. Thus a slow, low specificity enzyme can be effective. TEIIs from different pathways have differing specificities, but general trends include a preference for decarboxylated acyl units over carboxylated acyl units (5Heathcote M.L. Staunton J. Leadlay P.F. Chem. Biol. 2001; 8: 207-220Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 6Schwarzer D. Mootz H.D. Linne U. Marahiel M.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14083-14088Crossref PubMed Scopus (165) Google Scholar, 27Kim B.S. Cropp T.A. Beck B.J. Sherman D.H. Reynolds K.A. J. Biol. Chem. 2002; 277: 48028-48034Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), substrates linked to a carrier domain over substrates linked to CoA or the phosphopantetheine mimic N-acetylcysteamine (7Yeh E. Kohli R.M. Bruner S.D. Walsh C.T. ChemBioChem. 2004; 5: 1290-1293Crossref PubMed Scopus (86) Google Scholar, 28Tang Y. Koppisch A.T. Khosla C. Biochemistry. 2004; 43: 9546-9555Crossref PubMed Scopus (41) Google Scholar), and single amino acids over di- or tri-peptides (6Schwarzer D. Mootz H.D. Linne U. Marahiel M.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14083-14088Crossref PubMed Scopus (165) Google Scholar, 7Yeh E. Kohli R.M. Bruner S.D. Walsh C.T. ChemBioChem. 2004; 5: 1290-1293Crossref PubMed Scopus (86) Google Scholar). TEIIs are able to hydrolyze substrates attached to carrier domains from their native pathway as well as other pathways (6Schwarzer D. Mootz H.D. Linne U. Marahiel M.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14083-14088Crossref PubMed Scopus (165) Google Scholar, 20Hu Z. Pfeifer B.A. Chao E. Murli S. Kealey J. Carney J.R. Ashley G. Khosla C. Hutchinson C.R. Microbiology. 2003; 149: 2213-2225Crossref PubMed Scopus (42) Google Scholar, 28Tang Y. Koppisch A.T. Khosla C. Biochemistry. 2004; 43: 9546-9555Crossref PubMed Scopus (41) Google Scholar). PKS/NRPS/FAS thioesterases belong to the α/β hydrolase family. Structures are reported for seven PKS/NRPS/FAS thioesterases: crystal structures for the TEIs from the pikromycin (PikTE) PKS (29Tsai S.C. Lu H. Cane D.E. Khosla C. Stroud R.M. Biochemistry. 2002; 41: 12598-12606Crossref PubMed Scopus (100) Google Scholar), 6-deoxyerythronolide B (DEBSTE) PKS (30Tsai S.-C. Miercke L.J.W. Krucinski J. Gokhale R. Chen J.C.H. Foster P.G. Cane D.E. Khosla C. Stroud R.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14808-14813Crossref PubMed Scopus (172) Google Scholar), surfactin NRPS (SrfTE) (31Bruner S.D. Weber T. Kohli R.M. Schwarzer D. Marahiel M.A. Walsh C.T. Stubbs M.T. Structure. 2002; 10: 301-310Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), fengycin NRPS (FenTE) (32Samel S.A. Wagner B. Marahiel M.A. Essen L.-O. J. Mol. Biol. 2006; 359: 876-889Crossref PubMed Scopus (98) Google Scholar), and human fatty acid synthase (hFasTE) (33Chakravarty B. Gu Z. Chirala S.S. Wakil S.J. Quiocho F.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15567-15572Crossref PubMed Scopus (134) Google Scholar) systems, and NMR structures for enterobactin TEI (34Frueh D.P. Arthanari H. Koglin A. Vosburg D.A. Bennett A.E. Walsh C.T. Wagner G. Nature. 2008; 454: 903-906Crossref PubMed Scopus (130) Google Scholar), and surfactin TEII (35Koglin A. Lohr F. Bernhard F. Rogov V.V. Frueh D.P. Strieter E.R. Mofid M.R. Guntert P. Wagner G. Walsh C.T. Marahiel M.A. Dotsch V. Nature. 2008; 454: 907-911Crossref PubMed Scopus (105) Google Scholar). Like PKS modules, PKS TEIs are dimers. The dimer interface comprises two N-terminal helices that are unique to the PKS TEIs. NRPS TEIs are monomeric, like NRPS modules. The NRPS TEII of surfactin is also monomeric (31Bruner S.D. Weber T. Kohli R.M. Schwarzer D. Marahiel M.A. Walsh C.T. Stubbs M.T. Structure. 2002; 10: 301-310Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Although the FAS complex is dimeric, the FAS TEI is a monomer (33Chakravarty B. Gu Z. Chirala S.S. Wakil S.J. Quiocho F.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 15567-15572Crossref PubMed Scopus (134) Google Scholar). All of the TEs have an α-helical insertion after strand β5 that forms a lid over the active site. Additionally, in the PKS TEIs, the N-terminal dimer-forming helices contribute to the lid structure, forming a fixed channel that runs the length of the TE and contains the active site. In contrast, the active site pocket of monomeric NRPS TEIs and TEIIs is flexible; two conformations of the lid and active site pocket were observed in the surfactin TEI (SrfTEI) crystal structure (7Yeh E. Kohli R.M. Bruner S.D. Walsh C.T. ChemBioChem. 2004; 5: 1290-1293Crossref PubMed Scopus (86) Google Scholar), and chemical shift observations suggested greater flexibility for residues of the lid region in the surfactin TEII (SrfTEII) solution structure (35Koglin A. Lohr F. Bernhard F. Rogov V.V. Frueh D.P. Strieter E.R. Mofid M.R. Guntert P. Wagner G. Walsh C.T. Marahiel M.A. Dotsch V. Nature. 2008; 454: 907-911Crossref PubMed Scopus (105) Google Scholar). These movements seem to be of functional importance, because a movement of a linker peptide in SrfTEI determines the shape of the active site pocket and a movement of the first lid helix appears to modulate access to the active site (31Bruner S.D. Weber T. Kohli R.M. Schwarzer D. Marahiel M.A. Walsh C.T. Stubbs M.T. Structure. 2002; 10: 301-310Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). We report the structure and activity of recombinant RifR, the TEII of the rifamycin biosynthetic cluster. Steady-state kinetic analysis of the hydrolytic activity of RifR on a wide range of acyl-CoA and acyl-ACP substrates demonstrates that acyl-ACP substrates are preferred over the acyl-CoAs. Aberrant, decarboxylated acyl units are processed more efficiently than are the natural rifamycin building blocks. We report the crystal structure of RifR, the first for any hybrid PKS/NRPS TEII. The size and shape of the substrate chamber are variable, because one of the elements forming the chamber, an extended linker segment, is highly flexible, and different crystal forms reveal different shapes for the substrate binding site. Access to the active site is severely restricted, and structural comparisons with other thioesterases suggest that a conformational change in the lid and the flexible linker region is required for access to the substrate pocket. Materials—Nonradioactive acyl-CoAs were obtained from Sigma at the highest purity available. DL-2-[methyl-14C]Methylmalonyl-CoA (54 mCi/mmol) and [malonyl-2-14C]malonyl-CoA (52 mCi/mmol) were from PerkinElmer Life Sciences, and [acetyl-1-14C]acetyl-CoA (54 mCi/mmol) and [propionyl-1-14C]propionyl-CoA (53 mCi/mmol) were from Moravek Biochemicals. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and restriction enzymes were from Invitrogen. Manipulation of DNA and Strains—DNA manipulations were performed in Escherichia coli Novablue (Novagen) or DH5α using standard culture conditions (36Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Polymerase chain reactions were carried out using Platinum Pfx polymerase (Invitrogen) as recommended by the manufacturer. Construction of Expression Vectors for Wild-type and S94A RifR—PCR-based gene synthesis was used to assemble the rifR gene (GenBank™ accession number AF040570, nucleotides 96034–96813) encoding RifR from a set of 34 overlapping oligonucleotides (37Hoover D.M. Lubkowski J. Nucleic Acids Res. 2002; 30: e43Crossref PubMed Scopus (396) Google Scholar). The terminal 5′- and 3′-oligonucleotides were designed to flank the synthetic gene with NdeI and XhoI restriction sites, respectively. After assembly, the gene was PCR-amplified, digested with NdeI and XhoI, and ligated to pET21 (Novagen) digested with the same enzymes to generate pMS8, an expression vector for RifR with a natural N terminus and a hexahistidine sequence appended to its C terminus. The identity of the rifR synthetic gene was confirmed by DNA sequencing. The QuikChange method (Stratagene) was used to generate the S94A mutant of RifR; the serine nucleophile of the catalytic triad was converted to alanine by mutating AGT to GCT at the appropriate location in pMS8 to give expression vector pHC2. The mutation was confirmed by sequencing. Construction of an Expression Vector for S639A Rif M1—The natural sequence 5′-CGCGCC-3′ at nucleotides 24260–24265 (GenBank™ accession number AF040570), corresponding to the C-terminal end of Rif Module1 (M1), was chosen on the basis of an alignment of DEBS and Rif thiolation (T) domain sequences (38Gokhale R.S. Tsuji S.Y. Cane D.E. Khosla C. Science. 1999; 284: 482-485Crossref PubMed Scopus (294) Google Scholar) for replacement with the SpeI recognition sequence 5′-ACTAGT-3′. The BsaBI-SpeI fragment encoding Rif M1 was then fused to the SpeI-EcoRI fragment encoding the DEBS TE via replacement of the BsaBI-SpeI fragment encoding DEBS M3 in pST132 (39Tsuji S.Y. Cane D.E. Khosla C. Biochemistry. 2001; 40: 2326-2331Crossref PubMed Scopus (119) Google Scholar) to give pSA10. The presence of the DEBS TE domain was undesirable for this study, so its coding sequence was eliminated by ligating the NdeI-SpeI fragment of pSA10 encoding Rif M1 to the NdeI-NheI fragment of pET25b (Novagen). This yielded pMS24, an expression vector for Rif M1 with hexahistidine appended to the C terminus. The QuikChange method (Stratagene) was used to generate Rif M1 with an inactive acyltransferase domain: the active site serine of the acyltransferase domain was converted to alanine by mutating TCG at nucleotides 21434–21436 of the original sequence to GCG to give expression vector pMS25, which was fully sequenced to confirm its identity. Expression and Purification of Proteins—Expression plasmids were transformed into E. coli strain BL21 Star™ (DE3) (Invitrogen). One-liter cultures were grown at 37 °C in 2-liter flasks containing LB medium supplemented with 0.1 mg/ml ampicillin. Protein expression was induced with 100 μm isopropyl β-d-thiogalactopyranoside at an optical density at 600 nm of 0.8. After induction, incubation was continued for 20 h at 15 °C. The cells were then harvested by centrifugation at 2500 × g and resuspended in 50 mm sodium phosphate (pH 8.0), 300 mm NaCl, 10 mm imidazole, 1 mm MgCl2, 1 mm CaCl2, 0.1 mg/ml DNase I, 10% v/v glycerol. All purification procedures were performed at 4 °C. The resuspended cells were disrupted by two passages through a French press at 16,000 p.s.i., and the lysate was collected by centrifugation at 47,800 × g and loaded onto a previously equilibrated Histrap HP column (1 ml; GE Healthcare). The column was washed with 10 mm imidazole in 50 mm sodium phosphate (pH 8), 300 mm NaCl, 10% v/v glycerol, and the proteins were eluted with an imidazole gradient (10–100 mm) in the same solution. For Rif M1, pooled fractions containing S639A Rif M1 were diluted with 20 mm Tris (pH 7.5), 50 mm NaCl, 1 mm EDTA, 10% v/v glycerol and loaded onto a previously equilibrated HiTrapQ HP anion exchange column (1 ml; GE Biosciences). The column was washed with 50 mm NaCl in 20 mm Tris (pH 7.5), 1 mm EDTA, 10% v/v glycerol, and S639A Rif M1 was eluted with a NaCl gradient (50–500 mm) in the same solution. Pooled fractions containing S639A Rif M1 were buffer-exchanged into 50 mm HEPES (pH 7.5), 50 mm NaCl, 1 mm EDTA, 1 mm TCEP, 10% v/v glycerol and concentrated with an Amicon Ultra-15 centrifugal filter unit (Millipore). For wild-type and S94A RifR, metal affinity column fractions containing RifR were pooled, diluted with 20 mm Tris (pH 7.5), 50 mm NaCl, 1 mm EDTA, 10% v/v glycerol, and loaded onto a previously equilibrated Mono Q 5/50 GL anion exchange column (GE Biosciences). RifR was present in the column flow through and was buffer-exchanged into 50 mm HEPES (pH 7.5), 50 mm NaCl, 1 mm EDTA, 1 mm TCEP, 10% (v/v) glycerol and concentrated with an Amicon Ultra-15 centrifugal filter unit (Millipore). Purified proteins were flash-frozen in liquid nitrogen and stored at -80 °C. Protein concentrations were determined using the calculated extinction coefficients (40Gasteiger E. Hoogland C. Gattiker A. Durand S. Wilkins M.R. Appel R.D. Bairoch A. Walker J.M. The Proteomics Protocols Handbook. Humana Press, Totowa, NJ2005: 571-607Crossref Google Scholar) at 280 nm: 18,450 m-1 cm-1 for RifR, and 166,840 m-1 cm-1 for S639A Rif M1. Typical 1-liter cultures yielded 10 mg of purified RifR or 4 mg of purified S639A Rif M1. Selenomethionyl (SeMet) RifR was produced with a protocol as for RifR, modified according to Guerrero et al. (41Guerrero S.A. Hecht H.J. Hofmann B. Biebl H. Singh M. Appl. Microbiol. Biotechnol. 2001; 56: 718-723Crossref PubMed Scopus (85) Google Scholar), in which a 50-ml overnight culture was pelleted and added to minimal medium supplemented with SeMet prior to induction. Measurement of RifR Activity toward Acyl-CoA Substrates—Starting acyl-CoA stocks contained a small amount of CoA. Acyl-CoAs (25–1000 μm) were incubated with RifR or S94A RifR (2.5–25 μm) or no enzyme in the presence of 50 mm HEPES (pH 7.5), 25 mm NaCl, 5 mm MgCl2, 1 mm TCEP, 5% v/v glycerol at 25 °C. To ensure accurately measurable hydrolysis for all acyl-CoAs over the same time frame, slower hydrolyzing acyl-CoAs (acetyl-CoA, isobutyryl-CoA, hexanoyl-CoA, malonyl-CoA, and methylmalonyl-CoA (250–1000 μm)) were incubated with 25 μm RifR, and faster hydrolyzing acyl-CoAs (butyryl-CoA, octanoyl-CoA and propionyl-CoA (25–1000 μm)) were incubated with 2.5 μm RifR. Because of its limited solubility, decanoyl-CoA was incubated at a lower concentration (25–250 μm) with RifR (2.5 μm) than were the other faster hydrolyzing substrates. At each time point, aliquots were quenched to a final concentration of 5% trichloroacetic acid, and the precipitated protein was removed by centrifugation at 20,800 × g for 5 min. The ratio of acyl-CoA to CoA in the supernatant was quantified by HPLC using a C18 reverse phase column (Altima, 5 μm, 250 × 4.6 mm) monitored by absorbance at 259 nm. Separation was performed using a modification of a published protocol (42Deutsch J. Rapoport S.I. Rosenberger T.A. Neurochem. Res. 2002; 27: 1577-1582Crossref PubMed Scopus (24) Google Scholar) Briefly, a linear gradient of buffer A (75 mm potassium phosphate, pH 4.5) and buffer B (0.1% trifluoroacetic acid in acetonitrile) was used at a constant flow rate of 1.0 ml/min. Initial conditions were 96% buffer A and 4% buffer B. At 5 min, buffer B was increased to 7% over 5 min and then increased to 9% over 4 min. At 14 min, buffer B was increased to 50% over 5 min and maintained for 8 min. At 27 min, buffer B was decreased to 4% over 1 min, and the column was equilibrated at 4% buffer B for 8 min between injections. Retention times were as follows: acetyl-CoA, 18.6 min; butyryl-CoA, 20.5 min; CoA, 14.8 min; decanoyl-CoA, 22.6 min; hexanoyl-CoA, 21.4 min; isobutyryl-CoA, 20.5 min; malonyl-CoA, 13.5 min; methylmalonyl-CoA, 16.8 min; octanoyl-CoA, 22.0 min; and propionyl-CoA, 20.0 min. With the exception of isobutyryl-CoA, which was shown to saturate wild-type RifR, hydrolysis of acyl-CoAs was linearly dependent on enzyme concentration in the wild-type RifR reactions. No hydrolysis was detected in the control reactions without RifR, nor was hydrolysis observed in the S94A reactions except with isobutyryl-CoA and propionyl-CoA. Data analysis was performed using Kaleidagraph (Synergy Software). Initial velocities were extracted by fitting the hydrolysis progress plot to the equation: CoA fraction = 1 - (1 - CoA fraction0)e-tv0, where CoA fraction = [CoA]/([CoA] + [Acyl-CoA]), t = time, and v0 = initial velocity (Table 1).TABLE 1Kinetic parameters for RifR hydrolysis of acyl substratesSubstratekcat/KmRatioWild type RifRS94A RifRWild type/S94AACP/CoAm–1 s–1CoA substrates Decanoyl-CoACH3-(CH2)8-CO-S-CoA160 ± 18<0.16>1000 Octanoyl-CoACH3-(CH2)6-CO-S-CoA31 ± 2.5<0.16>190 Propionyl-CoACH3-CH2-CO-S-CoA25 ± 0.50.96 ± 0.3726 Butyryl-CoACH3-(CH2)2-CO-S-CoA13 ± 3.2<0.04>340 Acetyl-CoACH3-CO-S-CoA11 ± 0.2<0.03>320 Isobutyryl-CoA(CH3)2-CH-CO-S-CoA9.6 ± 0.084.5 ± 0.082.1 Hexanoyl-CoACH3-(CH2)4-CO-S-CoA5.9 ± 0.33<0.04>140 Methylmalonyl-CoACO2-(CH3)CH2-CO-S-CoA1.8 ± 0.17<0.03>72 Malonyl-CoACO2-CH2-CO-S-CoA1.5 ± 0.08<0.07>21ACP substrates Propionyl-RifM1CH3-CH2-CO-S-ACP210 ± 208.4 Acetyl-RifM1CH3-CO-S-ACP150 ± 3814 Methylmalonyl-RifM1CO2-(CH3)CH2-CO-S-ACP54 ± 6.330 Open table in a new tab Measurement of RifR Activity toward Acyl-S639A Rif M1 Substrates—To generate [14C]acyl-S639A Rif M1 substrates, [14C]acyl groups were installed on the apo T domain of S639A Rif M1 by preincubating the apo protein with [14C]acyl-CoA and the promiscuous phosphopantetheinyl transferase Sfp (43Lambalot R.H. Gehring A.M. Flugel R.S. Zuber P. LaCelle M. Marahiel M.A. Reid R. Khosla C. Walsh C.T. Chem. Biol. 1996; 3: 923-936Abstract Full Text PDF PubMed Scopus (725) Google Scholar, 44Quadri L.E. Weinreb P.H. Lei M. Nakano M.M. Zuber P. Walsh C.T. Biochemistry. 1998; 37: 1585-1595Crossref PubMed Scopus (541) Google Scholar). Preincubation reactions were performed at 25 °C for 60–90 min and contained 25 μm apo S639A Rif M1, 25 μm Sfp, and 25 μm [14C]acyl-CoA in 50 mm HEPES (pH 7.5), 25 mm NaCl, 5 mm MgCl2, 1 mm TCEP, 5% (v/v) glycerol. Aliquots of the preincubation reactions were then distributed into reaction tubes containing wild-type RifR, S94A RifR, or no TEII, for final reactions that consisted of varying concentrations of [14C]acyl-S639A Rif M1 (2–12 μm) and wild-type or S94A RifR (0–4 μm) in 50 mm HEPES (pH 7.5), 25 mm NaCl, 5 mm MgCl2, 1 mm TCEP, 5% v/v glycerol (plus residual Sfp and the 3′,5′-ADP product of preincubation reactions). Final reactions were incubated at 25 °C, and at desired time points 10-μl aliquots were quenched in an equal volume of 10% trichloroacetic acid. The protein precipitate was pelleted by centrifugation, washed with 150 μl of 5% trichloroacetic acid, and solubilized in 20 μl of 2% SDS, 50 mm Tris (pH 8). This solution was combined with 5 ml of liquid scintillation fluid (Ultima Gold; PerkinElmer Life Science), and the amount of [14C]acyl-S639A Rif M1 remaining at each time point was quantified by liquid scintillation counting. Disappearance of [14C]acyl-S639A Rif M1 substrates was linearly dependent on enzyme concentration in the wild-type RifR reactions, but little or no breakdown of [14C]acyl-S639A Rif M1 substrates was observed in the no-TEII and S94A RifR control reactions. Data analysis was performed using Kaleidagraph (Synergy Software), and exponential fits to the data typically gave R ≥ 0.95. To determine the identity of the acyl products of the RifR reactions, the trichloroacetic acid supernatants of late reaction time points were analyzed by radio-HPLC. The samples were injected onto a System Gold HPLC (Beckman) equipped with an Aminex HPX-87H ion exclusion column (Bio-Rad) and a Radiomatic 150TR flow scintillation analyzer (PerkinElmer Life Science) to separate and detect 14C-labeled species. Separations were performed isocratically in 0.008 n sulfuric acid over 30 min with a flow rate of 0.6 ml/min, and flow scintillation analysis was performed on the column eluant after it was mixed with Ultima Flo liquid scintillation fluid (PerkinElmer Life Science) in a 1 to 2 ratio. As expected, [14C]acetate, [14C]propionate, and [14C]methylmalonate predominated in the trichloroacetic acid supernatants from reactions that contained [14C]acetyl-S639A Rif M1, [14C]propionyl-S639A Rif M1, and [14C]methylmalonyl-S639A Rif M1, respectively. However, significant amounts of both [14C]malonate and [14C]acetate were detected in trichloroacetic acid supernatants from [14C]malonyl-S639A Rif M1 reactions; [14C]acetate presumably results from decarboxylation of [14C]malonyl-S639A Rif M1 to [14C]acetyl-S639A Rif M1 followed by RifR-catalyzed hydrolysis during the reaction period. The concurrent decarboxylation of [14C]malonyl-S639A Rif M1 prevented us from obtaining a reliable kcat/Km value for its hydrolysis by RifR, but the accumulation of [14C]malonate over time indicates that malonyl-S639A Rif M1 is indeed a substrate. Crystallization—RifR was crystallized by hanging drop vapor diffusion at 4 °C. Crystallization dro