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• W1969989079 abstract "KshAB (3-Ketosteroid 9α-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O2. Nevertheless, the reactivity of KshAB with O2 was low in the presence of 1,4-androstadiene-3,17-dione, with a kcat/KmO2 of 2450 ± 80 m–1 s–1. The crystallographic structure of KshA, determined to 2.3Å, revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroid-transforming enzyme of biotechnological and medical importance. KshAB (3-Ketosteroid 9α-hydroxylase) is a two-component Rieske oxygenase (RO) in the cholesterol catabolic pathway of Mycobacterium tuberculosis. Although the enzyme has been implicated in pathogenesis, it has largely been characterized by bioinformatics and molecular genetics. Purified KshB, the reductase component, was a monomeric protein containing a plant-type [2Fe-2S] cluster and FAD. KshA, the oxygenase, was a homotrimer containing a Rieske [2Fe-2S] cluster and mononuclear ferrous iron. Of two potential substrates, reconstituted KshAB had twice the specificity for 1,4-androstadiene-3,17-dione as for 4-androstene-3,17-dione. The transformation of both substrates was well coupled to the consumption of O2. Nevertheless, the reactivity of KshAB with O2 was low in the presence of 1,4-androstadiene-3,17-dione, with a kcat/KmO2 of 2450 ± 80 m–1 s–1. The crystallographic structure of KshA, determined to 2.3Å, revealed an overall fold and a head-to-tail subunit arrangement typical of ROs. The central fold of the catalytic domain lacks all insertions found in characterized ROs, consistent with a minimal and perhaps archetypical RO catalytic domain. The structure of KshA is further distinguished by a C-terminal helix, which stabilizes subunit interactions in the functional trimer. Finally, the substrate-binding pocket extends farther into KshA than in other ROs, consistent with the large steroid substrate, and the funnel accessing the active site is differently orientated. This study provides a solid basis for further studies of a key steroid-transforming enzyme of biotechnological and medical importance. Mycobacterium tuberculosis, arguably the world's most successful pathogen, infects one-third of the human population and has again become a global threat due in part to the emergence of extensively drug-resistant strains (XDR-TB) that are virtually untreatable with current medicines (1World Health Organization Anti-tuberculosis Drug Resistance in the World, Report No. 4. World Health Organization, Geneva, Switzerland2008: 14-21Google Scholar). Despite this alarming development, a surprising amount of the pathogen's physiology remains unknown. One recently discovered aspect of the physiology of M. tuberculosis is its cholesterol catabolic pathway (2van der Geize R. Yam K. Heuser T. Wilbrink M.H. Hara H. Anderton M.C. Sim E. Dijkhuizen L. Davies J.E. Mohn W.W. Eltis L.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1947-1952Crossref PubMed Scopus (406) Google Scholar). Studies of mutants in cholesterol uptake (3Pandey A.K. Sassetti C.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 4376-4380Crossref PubMed Scopus (689) Google Scholar) and degradation (4Yam K. D'Angelo I. Kalscheuer R. Zhu H. Wang J. Snieckus V. Ly L.H. Converse P.J. Jacobs W.R. Strynadka N. Eltis L.D. PLoS Pathog. 2009; (in press)PubMed Google Scholar) in various animal models have indicated that cholesterol catabolism is most important during the chronic phase of infection, although the latter study also provided evidence that it occurs from an early stage and contributes to dissemination of the pathogen in the host. Further study of cholesterol catabolism and the pathway enzymes are required to elucidate the precise role of cholesterol catabolism in infection.The cholesterol catabolic pathway of M. tuberculosis involves degradation of the branched alkyl side chain and the four-ringed steroid nucleus, as occurs in Rhodococcus jostii RHA1, a nonpathogenic, mycolic acid-producing actinomycete (2van der Geize R. Yam K. Heuser T. Wilbrink M.H. Hara H. Anderton M.C. Sim E. Dijkhuizen L. Davies J.E. Mohn W.W. Eltis L.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1947-1952Crossref PubMed Scopus (406) Google Scholar), although it is unclear whether the order of this degradation is obligatory. Side-chain degradation proceeds via a β-oxidative type process. Degradation of the steroid nucleus is initiated by 3β-hydroxysteroid dehydrogenase, resulting in the formation of 4-cholestene-3-one (5Yang X. Dubnau E. Smith I. Sampson N.S. Biochemistry. 2007; 46: 9058-9067Crossref PubMed Scopus (80) Google Scholar). The successive actions of KstD (3-ketosteroid-1Δ-dehydrogenase), which catalyzes the 1,2-desaturation of ring A, and KshAB (3-ketosteroid 9α-hydroxylase) (Fig. 1) lead to the opening of ring B with concomitant aromatization of ring A (6van der Geize R. Hessels G.I. van Gerwen R. van der Meijden P. Dijkhuizen L. Mol. Microbiol. 2002; 45: 1007-1018Crossref PubMed Scopus (97) Google Scholar, 7van der Geize R. Hessels G.I. van Gerwen R. Vrijbloed J.W. van Der Meijden P. Dijkhuizen L. Appl. Environ. Microbiol. 2000; 66: 2029-2036Crossref PubMed Scopus (104) Google Scholar, 8van der Geize R. Hessels G.I. Dijkhuizen L. Microbiology. 2002; 148: 3285-3292Crossref PubMed Scopus (78) Google Scholar, 9van der Geize R. Dijkhuizen L. Curr. Opin. Microbiol. 2004; 7: 255-261Crossref PubMed Scopus (193) Google Scholar). Ring A is subsequently hydroxylated to yield a catechol and then subject to meta-cleavage (2van der Geize R. Yam K. Heuser T. Wilbrink M.H. Hara H. Anderton M.C. Sim E. Dijkhuizen L. Davies J.E. Mohn W.W. Eltis L.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1947-1952Crossref PubMed Scopus (406) Google Scholar, 4Yam K. D'Angelo I. Kalscheuer R. Zhu H. Wang J. Snieckus V. Ly L.H. Converse P.J. Jacobs W.R. Strynadka N. Eltis L.D. PLoS Pathog. 2009; (in press)PubMed Google Scholar). Interestingly, M. tuberculosis appears to catabolize ring A completely to CO2 while incorporating the side chain carbon into its lipid pool (3Pandey A.K. Sassetti C.M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 4376-4380Crossref PubMed Scopus (689) Google Scholar). Finally, the metabolic fate of rings C and D is unclear in M. tuberculosis. Consistent with the role of cholesterol catabolism in pathogenesis, a transposon disruption mutant of kshA strongly attenuated the growth of the pathogen in interferon-γ-activated macrophages, conditions that mimic the immune response (10Rengarajan J. Bloom B.R. Rubin E.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 8327-8332Crossref PubMed Scopus (551) Google Scholar).KshAB of M. tuberculosis is predicted to be a Rieske-type oxygenase (RO) 6The abbreviations used are: RO, Rieske oxygenase; ADD, 1,4-androstadiene-3,17,-dione; AD, 4-androstene-3,17-dione; 9-OHAD, 9-hydroxy-4-androstene-3,17-dione; 9-OHADD, 9-hydroxy-1,4-androstadiene-3,17-dione; HSA, 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9, 17-dione; NDO9816-4, naphthalene dioxygenase from Pseudomonas sp. NCIB 9816-4; OMO86, 2-oxoquinoline 8-monooxygenase of P. putida 86; CARDOJ3, carbazole 1,9α-dioxygenase of Janthinobacterium sp. J3; PDODB01, phthalate dioxygenase of B. cepacia DB01; BPDOB-356, biphenyl dioxygenase of P. pnomenusa B-356; TADOmt-2, toluate dioxygenase of P. putida mt-2; FAS, ferrous ammonium sulfate; rmsd, positional root mean square deviation; HPLC, high pressure liquid chromatography. comprising a reductase (Rv3571; kshB) and an oxygenase (Rv3526; kshA) (2van der Geize R. Yam K. Heuser T. Wilbrink M.H. Hara H. Anderton M.C. Sim E. Dijkhuizen L. Davies J.E. Mohn W.W. Eltis L.D. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 1947-1952Crossref PubMed Scopus (406) Google Scholar). Sequence analyses indicate that KshB contains a plant-type [2Fe2S] cluster and a flavin prosthetic group, whereas KshA contains a Rieske-type [2Fe2S] cluster, coordinated by two histidine and two cysteine residues, and a mononuclear iron center, coordinated by two histidines and one aspartate. The latter metallocenter mediates the oxygenation reaction in ROs. Gene disruption studies in Rhodococcus erythropolis SQ1 (6van der Geize R. Hessels G.I. van Gerwen R. van der Meijden P. Dijkhuizen L. Mol. Microbiol. 2002; 45: 1007-1018Crossref PubMed Scopus (97) Google Scholar) and Mycobacterium smegmatis (11Andor A. Jekkel A. Hopwood D.A. Jeanplong F. Ilkoy E. Konya A. Kurucz I. Ambrus G. Appl. Environ. Microbiol. 2006; 72: 6554-6559Crossref PubMed Scopus (39) Google Scholar) have established that KshAB catalyzes the 9α-hydroxylation of 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) to 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) and 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (HSA), respectively. However, the substrate specificity of KshAB is unclear, as is the physiological sequence of the reactions catalyzed by this enzyme and KstD.ROs catalyze a range of reactions in which one or both atoms of O2 are incorporated into an organic substrate in a stereo- and regio-specific manner (12Ferraro D.J. Gakhar L. Ramaswamy S. Biochem. Biophys. Res. Commun. 2005; 338: 175-190Crossref PubMed Scopus (256) Google Scholar). The reaction cycle requires two reducing equivalents, which originate from NAD(P)H and which are transferred from the reductase to the oxygenase, sometimes via a ferredoxin (12Ferraro D.J. Gakhar L. Ramaswamy S. Biochem. Biophys. Res. Commun. 2005; 338: 175-190Crossref PubMed Scopus (256) Google Scholar, 13Kovaleva E.G. Lipscomb J.D. Nat. Chem. Biol. 2008; 4: 186-193Crossref PubMed Scopus (471) Google Scholar, 14Bugg T.D. Ramaswamy S. Curr. Opin. Chem. Biol. 2008; 12: 134-140Crossref PubMed Scopus (179) Google Scholar). The best characterized ROs, exemplified by naphthalene dioxygenase (NDO9816-4) from Pseudomonas sp. NCIB 9816–4 (15Kauppi B. Lee K. Carredano E. Parales R.E. Gibson D.T. Eklund H. Ramaswamy S. Structure. 1998; 6: 571-586Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 16Karlsson A. Parales J.V. Parales R.E. Gibson D.T. Eklund H. Ramaswamy S. Science. 2003; 299: 1039-1042Crossref PubMed Scopus (390) Google Scholar), catalyze the cis-dihydroxylation of aromatic compounds to initiate their aerobic catabolism by bacteria. These ring-hydroxylating dioxygenases have been studied extensively for their potential in bioremediation and as industrial biocatalysts (17Furukawa K. Trends Biotechnol. 2003; 21: 187-190Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 18Gibson D.T. Parales R.E. Curr. Opin. Biotechnol. 2000; 11: 236-243Crossref PubMed Scopus (490) Google Scholar). Structural studies have revealed that although the oxygenase component can comprise one or two different subunits, it is always trimeric: α3, α3β3, or(α3)2. In all cases, the α subunit comprises an N-terminal “Rieske” domain harboring the [2Fe-2S] cluster and a larger “catalytic” domain harboring the mononuclear iron (12Ferraro D.J. Gakhar L. Ramaswamy S. Biochem. Biophys. Res. Commun. 2005; 338: 175-190Crossref PubMed Scopus (256) Google Scholar). The residues coordinating these two metallocenters are conserved. Moreover, the α subunits are arranged head-to-tail within the trimer such that the [2Fe-2S] cluster and the mononuclear iron of adjacent subunits are with within ∼12 Å of each other and comprise the functional unit of the enzyme. An essential aspartate residue bridges the two metallocenters (19Parales R.E. Parales J.V. Gibson D.T. J. Bacteriol. 1999; 181: 1831-1837Crossref PubMed Google Scholar) and appears to play a role in preventing the activation of O2 in the absence of substrate (20Martins B.M. Svetlitchnaia T. Dobbek H. Structure. 2005; 13: 817-824Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Such an uncoupled reaction results in the futile consumption of NADH and production of reactive oxygen species, as has been observed in the presence of poor substrates (21Lee K. J. Bacteriol. 1999; 181: 2719-2725Crossref PubMed Google Scholar, 22Bernhardt F.H. Kuthan H. Eur. J. Biochem. 1981; 120: 547-555Crossref PubMed Scopus (22) Google Scholar, 23Imbeault N.Y. Powlowski J.B. Colbert C.L. Bolin J.T. Eltis L.D. J. Biol. Chem. 2000; 275: 12430-12437Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Among better characterized ROs, KshA shares greatest amino acid sequence identity (∼12%) with the oxygenases of 2-oxoquinoline 8-monooxygenase (OMO86) of Pseudomonas putida 86 (20Martins B.M. Svetlitchnaia T. Dobbek H. Structure. 2005; 13: 817-824Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and carbazole 1,9α-dioxygenase (CARDOJ3) of Janthinobacterium sp. strain J3 (24Nojiri H. Ashikawa Y. Noguchi H. Nam J.W. Urata M. Fujimoto Z. Uchimura H. Terada T. Nakamura S. Shimizu K. Yoshida T. Habe H. Omori T. J. Mol. Biol. 2005; 351: 355-370Crossref PubMed Scopus (74) Google Scholar), for which structural data are available, and phthalate dioxygenase (PDODB01) of Burkholderia cepacia DB01 (25Tarasev M. Ballou D.P. Biochemistry. 2005; 44: 6197-6207Crossref PubMed Scopus (38) Google Scholar), which has been well characterized kinetically.We describe herein the characterization of KshAB of M. tuberculosis. Each of the enzyme's two components was heterologously expressed, purified, and used to reconstitute the enzyme's activity. We investigated the substrate specificity of KshAB for two steroid metabolites as well as the enzyme's reactivity with O2. A crystal structure of KshA was solved, enabling structural comparisons with divergent ROs. The data are discussed in terms of the physiological role of KshAB as well as the structure and function of ROs.MATERIALS AND METHODSChemicals and Reagents-ADD was purchased from Steraloids, Inc. (Newport, RI). AD was purchased from Sigma. 2,3-Dihydroxybiphenyl was a gift from Dr. Victor Snieckus. Restriction enzymes and the Expand high fidelity PCR system were purchased from New England Biolabs (Ipswich, MA) and Roche Applied Science (Laval, Quebec, Canada), respectively. Oligonucleotides for amplifying kshA and kshB were purchased from the Nucleic Acid Protein Service Unit at the University of British Columbia. For kshA, the sequences were 5′-GCAATAGCATATGAGTACCGACACGAGTGGGGTCG-3′ and 5′-TCTAAGCTTTTGCTCGGCGGGCACGTCGT-3′. For kshB, the sequences were 5′-CGGAAGGCATATGACCGAGGCAATT-3′ and 5′-GACAAGCTTCTACTCGTCGTAGGTCACT-3′. Jeffamine M600 was purchased from Hampton Research (La Jolla, CA). All other reagents were of HPLC or analytical grade. Water for buffers was purified using a Barnstead Nanopure Diamond™ system (Dubuque, Iowa) to a resistance of at least 18 megaohms.DNA Manipulation and Plasmid Construction-DNA was propagated, digested, ligated, and transformed using standard protocols (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). DNA plasmids were purified as described previously (27Pulleyblank D. Michalak M. Daisley S.L. Glick R. Mol. Biol. Rep. 1983; 9: 191-195Crossref PubMed Scopus (25) Google Scholar) and were transformed into Escherichia coli by electroporation using a MicroPulser from Bio-Rad with Bio-Rad 0.1-cm GenePulser cuvettes. The kshA (Rv3526) and kshB (Rv3571) genes were amplified from M. tuberculosis H37Rv genomic DNA using polymerase chain reactions containing 0.2 μg of template DNA, 0.9 units of the Expand High Fidelity PCR System polymerase, a 50 μm concentration of each dNTP, and 75 pmol of each oligonucleotide in a volume of 25 μl. Reactions were subject to 25 temperature cycles using a Stratagene Robocycler Gradient 96 instrument (La Jolla, CA) as follows: 95 °C for 45 s, 45 °C for 45 s, and 72 °C for 90 s. The kshA and kshB amplicons were each digested with NdeI and HindIII and ligated into pET41b to yield pETKA1 and pETKB3, respectively. The kshA gene had its stop codon removed such that KshA was produced with a C-terminal His8 tag. Nucleotide sequences were confirmed by the Nucleic Acid Protein Service Unit at the University of British Columbia.Bacterial Strains and Growth-KshA and KshB were heterologously produced in E. coli BL21(DE3) using pETKA1 and pETKB3. Production of the two components was improved by producing them in cells that also contained pPAISC-1, a vector containing the iron sulfur cluster genes, as described previously (28Gomez-Gil L. Kumar P. Barriault D. Bolin J.T. Sylvestre M. Eltis L.D. J. Bacteriol. 2007; 189: 5705-5715Crossref PubMed Scopus (47) Google Scholar). Cells were grown in Luria broth supplemented with 25 μg/ml kanamycin, 7.5 μg/ml tetracycline, and an HCl-solubilized solution of minerals (29Vaillancourt F.H. Han S. Fortin P.D. Bolin J.T. Eltis L.D. J. Biol. Chem. 1998; 273: 34887-34895Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). One liter of medium inoculated with 10 ml of an overnight culture was incubated at 25 °C and 37 °C for KshA and KshB, respectively. When the OD reached 0.5, isopropyl 1-thio-β-d-galactopyranoside was added to a final concentration of 0.5 mm, and cultures were incubated for a further 18 h before harvesting by centrifugation. Pellets were washed twice with 20 mm sodium phosphate, pH 8.0, containing 10% glycerol and frozen at –80 °C until use.Protein Purification-Chromatography was performed using anÄKTA Explorer (Amersham Biosciences) unless otherwise stated. KshA was O2-labile and was therefore purified anaerobically by interfacing the system to a Labmaster model 100 glove box (M. Braun, Inc., Peabody, MA) operated at <5 ppm O2, as described previously (29Vaillancourt F.H. Han S. Fortin P.D. Bolin J.T. Eltis L.D. J. Biol. Chem. 1998; 273: 34887-34895Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). KshB was stable in air-saturated buffer and was therefore purified aerobically.To purify KshA, cells from 4 liters of culture were resuspended in 30 ml of 20 mm sodium phosphate, pH 8.0, containing 10% glycerol and disrupted by passing the suspension four times through an Emulsiflex-05 homogenizer (Avestin, Ottawa, Canada) operated at 10,000 p.s.i. Ferrous ammonium sulfate (FAS) was added to a final concentration of 0.25 mm after the first pass. The cell debris was removed by ultracentrifugation (10,000 × g for 45 min). The clear supernatant fluid (∼30 ml) was decanted and filtered through a 0.45-μm filter. This raw extract was loaded onto a gravity-operated Ni2+-nitrilotriacetic acid-agarose (Qiagen) column (1.8 × 4 cm) mounted in the glove box and equilibrated with 20 mm sodium phosphate buffer, pH 8.0, containing 10% glycerol. The protein was eluted using an imidazole step gradient according to the instructions of the manufacturer. The brown fraction eluted using 20 mm sodium phosphate buffer containing 150 mm imidazole, pH 8.0, and was exchanged into 25 mm HEPES, pH 7.5, containing 5% glycerol (buffer A) and concentrated to ∼7 ml using a stirred cell concentrator equipped with a YM30 membrane (Amicon, Oakville, Ontario). The solution was brought to 50 mm NaCl. Human α-thrombin (HTI, Essex Junction, VT) was then added to a molar ratio of 1:410 thrombin/KshA, and the solution was incubated at room temperature overnight. The sample was loaded onto a 1 × 10-cm column of Source™15Q (GE Healthcare) resin equilibrated with buffer A containing 1 mm dithiothreitol and 0.25 mm FAS. The enzyme was eluted with a step gradient of NaCl, with KshA eluting at 0.15 m NaCl. Brown-colored fractions were combined, exchanged into buffer A with 1 mm dithiothreitol and 0.25 mm FAS, concentrated to 20–25 mg/ml, and flash frozen as beads in liquid N2.To purify KshB, cells from 2 liters of culture were resuspended in buffer A, lysed, and filtered as described above, without adding ferrous ammonium sulfate to the lysate. The raw extract was loaded onto a 1 × 10-cm column Source™15Q resin equilibrated with buffer A. KshB was eluted with a linear gradient of 220–360 mm NaCl in 112 ml of buffer A. Orange-colored fractions were combined, exchanged into buffer A, concentrated to 20–25 mg/ml, and flash frozen in liquid N2. Purified KshA and KshB were stored at –80 °C. 2,3-Dihydroxybiphenyl dehydrogenase was prepared as previously described (29Vaillancourt F.H. Han S. Fortin P.D. Bolin J.T. Eltis L.D. J. Biol. Chem. 1998; 273: 34887-34895Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar).Analytical Methods-SDS-PAGE was performed using a Bio-Rad MiniPROTEAN III apparatus with a 12% resolving gel. Gels were stained with Coomassie Blue according to standard protocols. Protein concentrations were determined using the Micro BCA™ protein assay kit (Pierce) using bovine serum albumin as a standard. KshA concentrations were routinely determined using ∈280 = 142 mm–1 cm–1 and ∈324 = 23.2 mm–1 cm–1. Acid-labile sulfur content of samples was determined colorimetrically using the N,N-dimethyl-paraphenylene diamine assay (30Chen J.S. Mortenson L.E. Anal. Biochem. 1977; 79: 157-165Crossref PubMed Scopus (92) Google Scholar). Iron content was determined using the Ferene S assay (31Zabinski R. Munck E. Champion P.M. Wood J.M. Biochemistry. 1972; 11: 3212-3219Crossref PubMed Scopus (45) Google Scholar) adapted for the 96-well plate format. Briefly, 80-μl standards containing 5–75 μm FeCl2 and protein samples containing 1–2 nmol of iron were incubated for 10 min with 10 μl of 12 n HCl. Ten μl of 80% trichloroacetic acid was then added, and protein precipitate was removed by centrifugation. Supernatants were added to 20 μl of 45% sodium acetate in a 96-well plate, to which was then added 100 μl of Ferene S reagent (0.75 mm Ferene S, 10 mm l-ascorbic acid, 45% sodium acetate). Absorbances were read at 562 nm using a VMax kinetic microplate reader (Molecular Devices, Sunnyvale, CA). Prior to these analyses, KshA samples were exchanged into 0.1 m potassium phosphate at pH 7.0 by gel filtration chromatography to remove interfering substances. Gas chromatography-coupled mass spectrometry was performed using an HP 6890 series GC system fitted with an HP-5MS 30 m × 250 μm column (Hewlett-Packard, Palo Alto, CA) and an HP 5973 mass-selective detector.Kinetic Analysis-Enzyme activities were measured by following oxygen consumption using a Clarke-type electrode interfaced to a computer (model 5301; Yellow Springs Instruments, Yellow Springs, OH). The electrode was standardized using 2,3-dihydroxybiphenyl and 2,3-dihydroxybiphenyl dehydrogenase, and initial velocities were calculated from progress curves essentially as described previously (29Vaillancourt F.H. Han S. Fortin P.D. Bolin J.T. Eltis L.D. J. Biol. Chem. 1998; 273: 34887-34895Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The standard activity assay was performed in a total volume of 1.34 ml of air-saturated 0.1 m potassium phosphate, pH 7.0, containing 430 μm NADH, 380 μm ADD, 0.8 μm KshB, and 0.4 μm KshA. The reaction was initiated by adding KshA after equilibration of all other components for 30 s. Reaction velocities were corrected for oxygen consumption observed prior to KshA addition. Stock solutions were prepared fresh daily. KshA was thawed, exchanged into 0.1 m potassium phosphate, pH 7.0, anaerobically using gel filtration chromatography, and stored in a sealed vial on ice. Aliquots were withdrawn as required using gas-tight syringes. One unit of enzyme activity is defined as the amount of enzyme required to consume 1 μmol of substrate/min under the standard assay conditions.Apparent steady-state kinetic parameters for AD and ADD were determined by measuring rates of O2 consumption in the presence of various concentrations of substrate. Determinations of apparent steady-state kinetic parameters for O2 were measured in the presence of 380 μm ADD. In these experiments, the reaction buffer was equilibrated for 20 min by vigorous bubbling with mixtures of N2 and O2 prior to the reaction, and the reaction cuvette was continually flushed with the same gas mixture during the reaction. Kinetic parameters were evaluated by fitting the Michaelis-Menten equation to the data using the least-squares fitting and dynamic weighting options of LEONORA (32Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, Oxford1994Google Scholar).HPLC Analysis-Samples were analyzed using a Waters 2695 Separations HPLC module equipped with a Waters 2996 photodiode array detector and a 250 × 4.60-mm C18 Prodigy 10u ODS-Prep column (Phenominex, Torrance, CA). Flavins were identified using the method of Faeder and Siegel (33Faeder E.J. Siegel L.M. Anal. Biochem. 1973; 53: 332-336Crossref PubMed Scopus (221) Google Scholar). Briefly, 5 μl of 400 μm KshB was diluted to 500 μl using 0.1 m potassium phosphate, pH 7.7, containing 0.1 mm EDTA in a light-shielded tube. The mixture was heated at 100 °C for 3 min, rapidly cooled on ice, and then centrifuged at 10,000 × g for 30 min at 4 °C. The supernatant was passed through a 0.2-μm filter, and 100 μl was injected onto the HPLC column equilibrated with aqueous 0.5% phosphoric acid and operated at a flow rate of 1 ml/min. Flavins were eluted using a gradient of 0–100% methanol in 60 ml. Solutions of FMN and FAD were used as standards.Determination of enzyme coupling using ADD as a substrate was conducted in air-saturated 0.1 m potassium phosphate, pH 7.0, with 1 mm phenylalanine in the presence of 1.3 μm KshA and concentrations of all other components corresponding to those of the standard assay. Coupling experiments using AD as a substrate were performed as above with buffer equilibrated with 80% oxygen. 1 mm phenylalanine was not found to change the rate of reaction with either substrate (results not shown). After 5 min, reactions were quenched by diluting 200 μl of the reaction mixture in 200 μl of methanol. One hundred μl of this mixture was injected onto the column equilibrated with 30% methanol in 0.5% aqueous phosphoric acid. The column was operated at a flow rate of 1 ml/min, and the sample was eluted with a step gradient of methanol, with phenylalanine eluting with 30% methanol and ADD and AD eluting at 80% methanol. A standard curve of the peak area ratios of substrate and phenylalanine was used to quantify the substrate. Oxygen consumption was monitored using the oxygen electrode.Crystallization-Crystals of KshA were grown aerobically at room temperature (18 °C) using the sitting drop method. Drops contained a 1:1 ratio of 290–400 μm KshA in 25 mm HEPES, pH 7.0, with 1 mm dithiothreitol and 0.25 mm FAS and crystallization solution containing 1.1 m sodium malonate, 0.1 m HEPES, pH 7.0, and 0.5% (v/v) Jeffamine M600. Single dark brown crystals appeared in 3–5 weeks and grew to their full size (200 × 300 × 50 μm) in ∼3 months. Prior to data collection, KshA crystals were serially transferred between solutions of mother liquor supplemented with increasing amounts of ethylene glycol (5–20%) and flash frozen in liquid N2.X-ray Data Collection and Structure Determination-X-ray data collections were performed under cryogenic conditions using an in-house rotating anode x-ray generator (CuKa radiation λ = 1.542 Å) and at the Canadian Light Source (Beamline CMCF1, λ = 1.000 Å). Data were processed using XDS (34Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3214) Google Scholar) (Table 1). Substructure solution and initial phasing were performed using the PHENIX program suite (35Adams P.D. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. McCoy A.J. Moriarty N.W. Read R.J. Sacchettini J.C. Sauter N.K. Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1948-1954Crossref PubMed Scopus (3578) Google Scholar). The wavelength used (λ = 1.542 Å) and the redundant nature of the data allowed for the location of three iron and six sulfur sites using the HYSS (hybrid substructure search) heavy atom search routine, accounting for one molecule in the asymmetric unit. Initial single wavelength anomalous diffraction phasing was performed using PHASER (36McCoy A.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2007; 63: 32-41Crossref PubMed Scopus (1207) Google Scholar) (as implemented in PHENIX), enabling the calculation of a readily interpretable electron density map. The initial electron density map was then subjected to various cycles of density modification coupled to gradual phase extension and initial backbone tracing using the program Resolve (37Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 965-972Crossref PubMed Scopus (1632) Google Scholar). The resulting partial model was iteratively rebuilt using COOT (38Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (22822) Google Scholar), and the structure was refined using REFMAC (39Murshudov G.N. Vagin A.A." @default.
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