Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function
2008; Elsevier BV; Volume: 284; Issue: 1 Linguagem: Inglês
10.1074/jbc.m807003200
ISSN1083-351X
AutoresJeffrey M. Boyd, Jamie L. Sondelski, Diana M. Downs,
Tópico(s)Trace Elements in Health
ResumoThe ApbC protein has been shown previously to bind and rapidly transfer iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J., Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47, 8195–8202. This study utilized both in vivo and in vitro assays to examine the function of variant ApbC proteins. The in vivo assays assessed the ability of ApbC proteins to function in pathways with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S] cluster, and transfer [Fe-S] cluster. This study details the first kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of "deviant" Walker A proteins. Moreover, this study details the first functional analysis of mutant variants of the ever expanding family of ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that ApbC protein needs ATPase activity and the ability to bind and rapidly transfer [Fe-S] clusters for in vivo function. The ApbC protein has been shown previously to bind and rapidly transfer iron-sulfur ([Fe-S]) clusters to an apoprotein (Boyd, J. M., Pierik, A. J., Netz, D. J., Lill, R., and Downs, D. M. (2008) Biochemistry 47, 8195–8202. This study utilized both in vivo and in vitro assays to examine the function of variant ApbC proteins. The in vivo assays assessed the ability of ApbC proteins to function in pathways with low and high demand for [Fe-S] cluster proteins. Variant ApbC proteins were purified and assayed for the ability to hydrolyze ATP, bind [Fe-S] cluster, and transfer [Fe-S] cluster. This study details the first kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of "deviant" Walker A proteins. Moreover, this study details the first functional analysis of mutant variants of the ever expanding family of ApbC/Nbp35 [Fe-S] cluster biosynthetic proteins. The results herein show that ApbC protein needs ATPase activity and the ability to bind and rapidly transfer [Fe-S] clusters for in vivo function. Proteins containing iron-sulfur ([Fe-S]) clusters are employed in a wide array of metabolic functions (reviewed in Ref. 1Beinert H. J. Biol. Inorg. Chem. 2000; 5: 2-15Crossref PubMed Scopus (524) Google Scholar). Research addressing the biosynthesis of the iron-molybdenum cofactor of nitrogenase in Azotobacter vinelandii led to the discovery of an operon (iscAnifnifUSVcysE1) involved in the biosynthesis of [Fe-S] clusters (reviewed in Ref. 2Frazzon J. Dean D.R. Curr. Opin. Chem. Biol. 2003; 7: 166-173Crossref PubMed Scopus (187) Google Scholar). Subsequent experiments led to the finding of two more systems involved in the de novo biosynthesis of [Fe-S] clusters, the isc and the suf systems (3Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, 4Takahashi Y. Tokumoto U. J. Biol. Chem. 2002; 277: 28380-28393Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Like Escherichia coli, the genome of Salmonella enterica serovar Typhimurium encodes for the isc and suf [Fe-S] cluster biosynthesis machinery. Recent studies have identified a number of additional or non-isc/-suf-encoded proteins that are involved in bacterial [Fe-S] cluster biosynthesis and repair. Examples include the following: CyaY, an iron-binding protein believed to be involved in iron trafficking and iron delivery (5Layer G. Ollagnier-de Choudens S. Sanakis Y. Fontecave M. J. Biol. Chem. 2006; 281: 16256-16263Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 6Vivas E. Skovran E. Downs D.M. J. Bacteriol. 2006; 188: 1175-1179Crossref PubMed Scopus (48) Google Scholar, 7Bou-Abdallah F. Adinolfi S. Pastore A. Laue T.M. Dennis Chasteen N. J. Mol. Biol. 2004; 341: 605-615Crossref PubMed Scopus (110) Google Scholar); YggX, an Fe2+-binding protein that protects the cell from oxidative stress (8Cui Q. Thorgersen M.P. Westler W.M. Markley J.L. Downs D.M. Proteins Struct. Funct. Bioinformat. 2006; 62: 578-586Crossref PubMed Scopus (12) Google Scholar, 9Gralnick J.A. Downs D.M. J. Biol. Chem. 2003; 278: 20708-20715Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar); ErpA, an alternate A-type [Fe-S] cluster scaffolding protein (10Loiseau L. Gerez C. Bekker M. Ollagnier-de Choudens S. Py B. Sanakis Y. Teixeira de Mattos J. Fontecave M. Barras F. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 13626-13631Crossref PubMed Scopus (119) Google Scholar); NfuA, a proposed intermediate [Fe-S] delivery protein (11Bandyopadhyay S. Naik S.G. O'Carroll I.P. Huynh B.H. Dean D.R. Johnson M.K. Dos Santos P.C. J. Biol. Chem. 2008; 283: 14092-14099Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 12Angelini S. Gerez C. Ollagnier-de Choudens S. Sanakis Y. Fontecave M. Barras F. Py B. J. Biol. Chem. 2008; 283: 14084-14091Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 13Jin Z. Heinnickel M. Krebs C. Shen G. Golbeck J.H. Bryant D.A. J. Biol. Chem. 2008; 283: 28426-28435Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar); YtfE, a protein proposed to be involved in [Fe-S] cluster repair (14Justino M.C. Almeida C.C. Goncalves V.L. Teixeira M. Saraiva L.M. FEMS Microbiol. Lett. 2006; 257: 278-284Crossref PubMed Scopus (66) Google Scholar, 15Justino M.C. Almeida C.C. Teixeira M. Saraiva L.M. J. Biol. Chem. 2007; 282: 10352-10359Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar); and CsdA-CsdE, an alternative cysteine desulferase (16Loiseau L. Ollagnier-de Choudens S. Lascoux D. Forest E. Fontecave M. Barras F. J. Biol. Chem. 2005; 280: 26760-26769Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Analysis of the metabolic network anchored to thiamine biosynthesis in S. enterica identified lesions in three non-isc or -suf loci that compromise Fe-S metabolism as follows: apbC, apbE, and rseC (17Skovran E. Downs D.M. J. Bacteriol. 2003; 185: 98-106Crossref PubMed Scopus (61) Google Scholar, 18Petersen L. Downs D.M. J. Bacteriol. 1996; 178: 5676-5682Crossref PubMed Google Scholar, 19Koo M.S. Lee J.H. Rah S.Y. Yeo W.S. Lee J.W. Lee K.L. Koh Y.S. Kang S.O. Roe J.H. EMBO J. 2003; 22: 2614-2622Crossref PubMed Scopus (144) Google Scholar, 20Beck B.J. Connolly L.E. De Las Penas A. Downs D.M. J. Bacteriol. 1997; 179: 6504-6508Crossref PubMed Google Scholar, 21Beck B.J. Downs D.M. J. Bacteriol. 1998; 180: 885-891Crossref PubMed Google Scholar). This metabolic system was subsequently used to dissect a role for cyaY and gshA in [Fe-S] cluster metabolism (6Vivas E. Skovran E. Downs D.M. J. Bacteriol. 2006; 188: 1175-1179Crossref PubMed Scopus (48) Google Scholar, 22Gralnick J. Webb E. Beck B. Downs D. J. Bacteriol. 2000; 182: 5180-5187Crossref PubMed Scopus (33) Google Scholar, 23Skovran E. Lauhon C.T. Downs D.M. J. Bacteriol. 2004; 186: 7626-7634Crossref PubMed Scopus (52) Google Scholar). Of these, the apbC (mrp in E. coli) locus was identified as the predominant site of lesions that altered thiamine synthesis by disrupting [Fe-S] cluster metabolism (17Skovran E. Downs D.M. J. Bacteriol. 2003; 185: 98-106Crossref PubMed Scopus (61) Google Scholar, 18Petersen L. Downs D.M. J. Bacteriol. 1996; 178: 5676-5682Crossref PubMed Google Scholar). ApbC is a member of the ParA subfamily of proteins that have a wide array of functions, including electron transfer (24Igarashi R.Y. Seefeldt L.C. Crit. Rev. Biochem. Mol. Biol. 2003; 38: 351-384Crossref PubMed Scopus (205) Google Scholar), initiation of cell division (25Lutkenhaus J. Sundaramoorthy M. Mol. Microbiol. 2003; 48: 295-303Crossref PubMed Scopus (95) Google Scholar), and DNA segregation (26Leonard T.A. Butler P.J. Lowe J. EMBO J. 2005; 24: 270-282Crossref PubMed Scopus (227) Google Scholar, 27Friedman S.A. Austin S.J. Plasmid. 1988; 19: 103-112Crossref PubMed Scopus (97) Google Scholar). Importantly, ATP hydrolysis is required for function of all well characterized members of this subfamily, and all members contain a "deviant" Walker A motif, which contains two lysine residues instead of one (GKXXXGK(S/T)) (28Koonin E.V. J. Mol. Biol. 1993; 229: 1165-1174Crossref PubMed Scopus (259) Google Scholar). ApbC has been shown to hydrolyze ATP (17Skovran E. Downs D.M. J. Bacteriol. 2003; 185: 98-106Crossref PubMed Scopus (61) Google Scholar). Recently, five proteins with a high degree of identity to ApbC have been shown to be involved in [Fe-S] cluster metabolism in eukaryotes. The sequence alignments of the central portion of these proteins and bacterial ApbC are shown in Fig. 1. HCF101 was demonstrated to be involved in chloroplast [Fe-S] cluster metabolism (29Lezhneva L. Amann K. Meurer J. Plant J. 2004; 37: 174-185Crossref PubMed Scopus (109) Google Scholar, 30Stockel J. Oelmuller R. J. Biol. Chem. 2004; 279: 10243-10251Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The CFD1, Npb35, and huNbp35 (formally Nubp1) proteins were demonstrated to be involved in cytoplasmic [Fe-S] cluster metabolism (31Roy A. Solodovnikova N. Nicholson T. Antholine W. Walden W.E. EMBO J. 2003; 22: 4826-4835Crossref PubMed Scopus (143) Google Scholar, 32Hausmann A. Aguilar Netz D.J. Balk J. Pierik A.J. Muhlenhoff U. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3266-3271Crossref PubMed Scopus (134) Google Scholar). Ind1 was demonstrated to be involved in the maturation of [Fe-S] clusters in the mitochondrial enzyme NADH:ubiquinone oxidoreductase (33Bych K. Kerscher S. Netz D.J. Pierik A.J. Zwicker K. Huynen M.A. Lill R. Brandt U. Balk J. EMBO J. 2008; 27: 1736-1746Crossref PubMed Scopus (143) Google Scholar). There is currently no report of any of these proteins hydrolyzing ATP. Biochemical analysis of ApbC indicated that it could bind and transfer [Fe-S] clusters to Saccharomyces cerevisiae apo-isopropylmalate isomerase (34Boyd J.M. Pierik A.J. Netz D.J. Lill R. Downs D.M. Biochemistry. 2008; 47: 8195-8202Crossref PubMed Scopus (46) Google Scholar). Additional genetic studies indicated that ApbC has a degree of functional redundancy with IscU, a known [Fe-S] cluster scaffolding protein (35Boyd J.M. Lewis J.A. Escalante-Semerena J.C. Downs D.M. J. Bacteriol. 2008; 190: 4596-4602Crossref PubMed Scopus (25) Google Scholar, 36Raulfs E.C. O'Carroll I.P. Dos Santos P.C. Unciuleac M.C. Dean D.R. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 8591-8596Crossref PubMed Scopus (110) Google Scholar). In this study we investigate the correlation between the biochemical properties of ApbC (i.e. ATPase activity, [Fe-S] cluster binding, and [Fe-S] cluster transfer rates) and the in vivo function of this protein. This is the first detailed kinetic analysis of ATP hydrolysis for a member of the ParA subfamily of deviant Walker A proteins and the first functional analysis of a member of the ever expanding family of ApbC/Nbp35 proteins. Data presented indicate that noncomplementing variants have distinct biochemical properties that place them in three distinct classes. Materials—FeCl3 (ACS grade), thiamine (>99%), Li2S (>98%), Fe(NH4)2(SO4)2 (>99%), 3-(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5′,5″-disulfonic acid (>99%), ascorbic acid (>99%), (C2H3O2)2Zn·2H2O (reagent grade), N,N-dimethyl-p-phenylenediamine sulfate (98%), and l-cysteine (>98%) were purchased from Sigma. dl-threo-3-Isopropylmalic acid (96%) was purchased from Wako Pure Chemical Co Osaka, Japan. All other chemicals were of the highest purity available. The BCA protein assay kit and bovine serum albumin were purchased from Pierce. E. coli BL21 (AI*) cells were purchased from Novagen. Bacterial Strains, Culture Media, and Growth Conditions—Bacterial strains were derivatives of S. enterica serovar Typhimurium strain LT2 (specifically DM1) whose genotypes are shown in Table 1. Plasmids carrying the desired allele of apbC fused to a C-terminal His tag in pET20b were constructed as described (17Skovran E. Downs D.M. J. Bacteriol. 2003; 185: 98-106Crossref PubMed Scopus (61) Google Scholar) using XhoI instead of NcoI. All constructs used were verified by sequencing at the University of Wisconsin Biotechnology Center. Plasmids were moved between strains via electroporation. Overnight cultures were grown in Difco nutrient broth with appropriate antibiotic for strains harboring a plasmid. Test cultures were grown at 37 °C shaking at an approximate 45° angle to increase aeration. The absorbance of cultures was taken at 650 nm on a Bausch and Lomb Spectronic 20.TABLE 1Strains and plasmidsStrainGenotypeDM5104DM1 wild typeDM5986apbC55:Tn10d(Tc) yggX::GmaResistances are indicated as follows: Tc, tetracycline; Ap, ampicillin; Gm, gentamycin.DM9450purF2085 apbC55::Tn10d(Tc)PlasmidVector and insertInsertpET20bpET20b(Ap)NonepES1pET20b(Ap)apbCbPlasmids were constructed for a previous study (17).pES2pET20b(Ap)apbC (K121A)bPlasmids were constructed for a previous study (17).pJB1pET20b(Ap)apbC (S122A)pJB2pET20b(Ap)apbC (C70A)pJB3pET20b(Ap)apbC (C283A)pJB4pET20b(Ap)apbC (C286A)pJB5pET20b(Ap)apbC (S182A)pJB6pET20b(Ap)apbC (S116A)pJB7pET20b(Ap)apbC (C283A, C286A)a Resistances are indicated as follows: Tc, tetracycline; Ap, ampicillin; Gm, gentamycin.b Plasmids were constructed for a previous study (17Skovran E. Downs D.M. J. Bacteriol. 2003; 185: 98-106Crossref PubMed Scopus (61) Google Scholar). Open table in a new tab Nutritional Analysis—Complementation analyses were performed in strains where plasmids encoded the only copy of apbC present in the cell. Complementation was assessed using 5-ml cultures of no carbon E (NCE) medium supplemented with 1 mm MgSO4 and trace minerals. Cultures were started with a 1:100 inoculum. When present in the media, the following were used at the specified final concentration: adenine, 0.4 mm; thiamine, 100 nm; glucose, 11 mm; tricarballylate, 20 mm; gluconate, 11 mm; and ampicillin, 30 μg/ml. Three growth assays, using two genetic backgrounds, were used to examine the ability of ApbC proteins to function in vivo. The two genetic backgrounds used were apbC yggX and purF apbC. In vivo function was assayed by growth on tricarballylate as a carbon and energy source or growth in the absence of exogenous thiamine on either glucose or gluconate, respectively. The requirement for ApbC in these growth conditions has been described previously (17Skovran E. Downs D.M. J. Bacteriol. 2003; 185: 98-106Crossref PubMed Scopus (61) Google Scholar, 18Petersen L. Downs D.M. J. Bacteriol. 1996; 178: 5676-5682Crossref PubMed Google Scholar, 23Skovran E. Lauhon C.T. Downs D.M. J. Bacteriol. 2004; 186: 7626-7634Crossref PubMed Scopus (52) Google Scholar, 35Boyd J.M. Lewis J.A. Escalante-Semerena J.C. Downs D.M. J. Bacteriol. 2008; 190: 4596-4602Crossref PubMed Scopus (25) Google Scholar, 37Lewis J.A. Horswill A.R. Schwem B.E. Escalante-Semerena J.C. J. Bacteriol. 2004; 186: 1629-1637Crossref PubMed Scopus (31) Google Scholar). Anaerobic Work—Anaerobic work was performed using a Coy anaerobic glove box (Grass Lake, MI) or vacuum manifold. Before placement inside the anaerobic chamber, solutions were made anoxic by repeated evacuation and flushing with N2 gas passed over a heated copper column for removal of O2. Outside of the glove box, all solutions were added to anaerobic cuvettes using gas-tight Hamilton syringes. ApbC and Leu-1 Protein Purification—Purification of ApbC and Leu-1 has been described previously (34Boyd J.M. Pierik A.J. Netz D.J. Lill R. Downs D.M. Biochemistry. 2008; 47: 8195-8202Crossref PubMed Scopus (46) Google Scholar). Protein Concentration Determination—Protein concentration was determined using a colorimetric assay or an empirically determined extinction coefficient (apo-ApbC-His280 = 43.1 mm–1 cm–1). The colorimetric assay was copper-based and used a reagent containing bicinchoninic acid to detect of the cupreous ion (Pierce). Bovine serum albumin (2 mg/ml) was used as a standard. [Fe-S] Cluster Reconstitution—Iron-sulfur cluster reconstitution was described previously (34Boyd J.M. Pierik A.J. Netz D.J. Lill R. Downs D.M. Biochemistry. 2008; 47: 8195-8202Crossref PubMed Scopus (46) Google Scholar). Briefly, ApbC protein (2.1 mg/ml; 52 μm) suspended in buffer A (50 mm Tris, pH 8.0, 150 mm NaCl) was incubated for 1 h in an anoxic environment in the presence of 5 mm DTT. 2The abbreviation used is: DTT, dithiothreitol. A 5-fold excess of FeCl3 was added to the protein followed by a 5-fold excess Li2S. Proteins were incubated for 1 h, and excess S2–, Fe3+, and DTT was removed by passing the reaction mixture over a PD-10 column (GE Healthcare). One mm DTT was added to protein post desalting. Leu-1 Activation Assays—Leu-1 activation assays have been described previously (34Boyd J.M. Pierik A.J. Netz D.J. Lill R. Downs D.M. Biochemistry. 2008; 47: 8195-8202Crossref PubMed Scopus (46) Google Scholar). Briefly, apo-isopropylmalate isomerase (apo-Leu-1) (25 μm) was pre-reduced with 5 mm DTT in the anaerobic glove box for at least 1 h prior to assay initiation. The Leu-1 activation assays contained 3.5 μm Leu-1, 5 mm DTT, 50 mm Tris-HCl, pH 8.0, and 150 mm NaCl in a total volume of 500 μl. Assays were initiated by the addition of 4 μm ApbC protein. Ten-μl aliquots of the assay mixture were removed at time points, and Leu-1 was assayed for the ability to convert 3-isopropylmalate to dimethylcitraconate acid spectrophotometrically (dimethylcitraconate ε235 = 4.35 mm–1 cm–1) (38Gross S.R. Burns R.O. Umbarger H.E. Biochemistry. 1963; 2: 1046-1052Crossref PubMed Scopus (36) Google Scholar). The second-order rate constant for ApbC dependent Leu-1 activation was determined by linearization of the data and fitting the data using linear regression. The ApbC-dependent activation of Leu-1 is summarized by Reaction 1,holo−ApbC+apo−Leu−1→holo−Leu−1Reaction 1 The amount of holo-ApbC protein present (4 μm initial) in the assay mixture at a given time point was determined using Equation 1,[holo−ApbC]=4−(SAx/SA40)ċ4Eq. 1 where SAx is the specific activity of Leu-1 at a fixed time point, and SA40 is the specific activity of Leu-1 after the reaction had proceeded for 40 min. The second-order rate constant of cluster transfer was determined by fitting the 1/[holo-ApbC protein] versus time post the addition of holo-ApbC protein to apo-Leu-1 data using linear regression, where k is the slope of the fit. Spectroscopic Techniques—The UV-visible absorption spectra were recorded with a Lambda Bio 40 spectrophotometer (PerkinElmer Life Sciences) using 1.5-ml Sterna Cell cuvettes that can be anaerobically sealed (Atascadero, CA). Metal Analysis—Inductively coupled plasma-mass spectrometry was conducted by the Soil and Plant Analysis Laboratory at the University of Wisconsin-Madison. The concentration of non-heme iron and acid-labile sulfide was also determined as described elsewhere (39Balk J. Pierik A.J. Netz D.J. Muhlenhoff U. Lill R. EMBO J. 2004; 23: 2105-2115Crossref PubMed Scopus (179) Google Scholar). Assay to Monitor Inorganic Phosphate—The direct colorimetric malachite green/molybdate/polyvinyl alcohol assay as described by Chan et al. (40Chan K.M. Delfert D. Junger K.D. Anal. Biochem. 1986; 157: 375-380Crossref PubMed Scopus (823) Google Scholar) was used to monitor inorganic phosphate (Pi) release from nucleotide hydrolysis. Assays contained 0–0.34 mg of purified wild-type ApbC, 0 or 5 mm MgCl2, 0 or 200 mm KCl, 0 or 5 mm nucleotide triphosphate, and 50 mm Tris, pH 8.0. The assays were initiated by addition of ApbC. The mixture was allowed to react at room temperature for 30 min before 20 μl of the reaction mixture was removed, diluted to 250 μl with buffer A, and quickly added to 1 ml of the malachite green/molybdate/polyvinyl alcohol assay mixture. The mixture was incubated for 15 min at room temperature before sample absorbance was monitored at 630 nm in a Molecular Devices Spectra-MAX Plus microplate reader. Because of daily variation of absorbance, a standard curve containing 0–15 nmol of Pi was constructed for each set of assays. Coupled Spectrophotometric Assay for ApbC ATPase Activity—A continuous spectrophotometric assay was developed that couples ADP production with the oxidation of NADH to NAD+ (41Kreuzer K.N. Jongeneel C.V. Methods Enzymol. 1983; 100: 144-160Crossref PubMed Scopus (105) Google Scholar) as shown in Reactions 2 and 3.ADP+phosphoenolpyruvate→ATP+pyruvateReaction 2 pyruvate+NADH+H+→NAD++lactateReaction 3 The assays were initiated when purified ApbC protein (10–350 μg) was added to an assay mixture containing 0.2 mm NADH, 4 mm phosphoenolpyruvate, 0–12 mm MgCl2, 200 mm KCl, 20 units of lactate dehydrogenase, 20 units of pyruvate kinase, 0.01–10 mm ATP (stocks prepared in 1 m Tris, pH 8.0), and 50 mm Tris, pH 8.0. The assay volume was 1 ml, and assays were conducted anaerobically in 1.5 ml of Sterna Cell cuvettes (Atascadero, CA) that had a screw cap top and rubber septum. After the addition of ApbC, the oxidation of NADH was monitored by following the absorbance change at A340 from 2 to 4 min (linear region). The linear steady-state portion of the A340 versus time plot was used to determine the Hill coefficient by fitting initial velocity data to Equation 2 (R2 ≥ 0.99),v=VmaxċS∧h/(Km∧h+S∧h)Eq. 2 where v is velocity; S is the concentration of varied substrate; Vmax is the velocity at saturating substrate; Km is the Michaelis constant for the varied substrate, and h is the Hill coefficient (labeled nH in Table 4). The kinetic parameters Vmax and Km were determined by fitting the v versus [ATP] initial velocity data to Equation 3 (R2 ≥ 0.99),v=Vmax1ċS/(Km1+S)+Vmax2ċS/(Km2+S)Eq. 3 The velocity data as plotted in the double-reciprocal form was fit by Equation 4,y=(a+bx+cx∧2)/(1+ex)Eq. 4 where the initial slope is defined as b – ae; the slope of the asymptote is c/e; the y intercept is a, and the asymptote intercept is (be – c)/e^2.TABLE 4Nucleotide specificity of ApbCNucleotide (5 mm)Pi releasenmol mg-1ATP518 ± 18ATP - Mg2+NDaNo phosphate release was detected.ATP - K+63 ± 27ATP + AMP429 ± 36ATP + ADPNDADPNDAMPNDdATP261 ± 30CTP42 ± 21GTP75 ± 15TTPNDUTPNDa No phosphate release was detected. Open table in a new tab Computational Analysis—Nonlinear and linear regression analyses and curve fitting to first-order rate laws were performed using the software SigmaPlot (version 9.0). Bioinformatic Analysis—Protein sequences were acquired from the NCBI website in FASTA format. The accession numbers for sequences used are as follows: Ind1 EAK91699; Nbp35 CAA96797.1; Cfd1, AAS56623; Hcf101, AAR97892.1, ApbC (mrp), NP_461098; and Nubp1, NP_002475.2. Sequence alignments were performed using the Clustal_W function of the Lasergene software by DNAstar, Madison, WI. Default settings were used, including the Gonnet Series for Protein Weight Matrix, gap penalty of 10, and a gap length penalty of 0.2. Selected regions of sequences were used to give the best alignment of the similar regions. The protein sequences for the proteins used in Fig. 1 were acquired in FASTA format from the NCBI Protein Sequence Data Base. The ApbC amino acid sequence was used as the input for the three-dimensional Jigsaw version 2.0 Comparative Modeling Server by Cancer Research UK using the automatic mode (42Bates P.A. Kelley L.A. MacCallum R.M. Sternberg M.J. Proteins. 2001; 5: S39-S46Crossref PubMed Scopus (489) Google Scholar, 43Bates P.A. Sternberg M.J. Proteins. 1999; 3: S47-S54Crossref PubMed Scopus (148) Google Scholar, 44Contreras-Moreira B. Bates P.A. Bioinformatics (Oxf.). 2002; 18: 1141-1142Crossref PubMed Scopus (142) Google Scholar). The resulting text file alignment was viewed using the PyMOL Viewer from Delano Scientific, LLC, for protein structures. Regions of the proteins that do not have significant similarity are excluded from the structures. The accession numbers of the structures used to thread the ApbC sequence are 2bej_A for Soj and 2afk_H for NifH and can be found on the NCBI website. Protein Sequence Alignments Show Significant Conservation of Protein Sequence within the ApbC/Nbp35 Subfamily of ParA Homologs—The ApbC/Nbp35 subfamily proteins show a high degree of conservation in their central and C-terminal domains. Representative members of the ApbC/Nbp35 family are shown in Fig. 1. The N-terminal 100 amino acids of bacterial ApbC are sparsely conserved in the plant homolog HCF101, yeast Nbp35, human Nubp1, and mitochondrial Ind1 and therefore are not included in the alignments. The N-terminal extension is missing in yeast Cfd1. The N-terminal region of ApbC showed no similarity to characterized functional domains of the N-terminal extension found in ParA and ParA homologs (25Lutkenhaus J. Sundaramoorthy M. Mol. Microbiol. 2003; 48: 295-303Crossref PubMed Scopus (95) Google Scholar). The central and C-terminal domains of these proteins contain two highly conserved domains as follows: the Walker A box for ATP binding/hydrolysis, and a CXXC motif in the C-terminal third of the protein. In Fig. 1 the Walker A box, conserved Cys residues, and conserved Ser are boxed for clarity. All six of these proteins are thought to be involved in [Fe-S] cluster metabolism (29Lezhneva L. Amann K. Meurer J. Plant J. 2004; 37: 174-185Crossref PubMed Scopus (109) Google Scholar, 30Stockel J. Oelmuller R. J. Biol. Chem. 2004; 279: 10243-10251Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 31Roy A. Solodovnikova N. Nicholson T. Antholine W. Walden W.E. EMBO J. 2003; 22: 4826-4835Crossref PubMed Scopus (143) Google Scholar, 32Hausmann A. Aguilar Netz D.J. Balk J. Pierik A.J. Muhlenhoff U. Lill R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3266-3271Crossref PubMed Scopus (134) Google Scholar, 33Bych K. Kerscher S. Netz D.J. Pierik A.J. Zwicker K. Huynen M.A. Lill R. Brandt U. Balk J. EMBO J. 2008; 27: 1736-1746Crossref PubMed Scopus (143) Google Scholar, 34Boyd J.M. Pierik A.J. Netz D.J. Lill R. Downs D.M. Biochemistry. 2008; 47: 8195-8202Crossref PubMed Scopus (46) Google Scholar, 35Boyd J.M. Lewis J.A. Escalante-Semerena J.C. Downs D.M. J. Bacteriol. 2008; 190: 4596-4602Crossref PubMed Scopus (25) Google Scholar). In Vivo Function of ApbC Is Abolished by Mutations in the Walker A Motif, CXXC Motif, and a Conserved Serine—A number of mutant alleles of apbC were created using plasmid pES1, which encodes the C-terminal hexahistidine-tagged ApbC protein (17Skovran E. Downs D.M. J. Bacteriol. 2003; 185: 98-106Crossref PubMed Scopus (61) Google Scholar). Directed mutations substituted single residues to alanine in three regions of the protein (see Fig. 1) as follows: 1) deviant Walker A motif necessary for ATP binding/hydrolysis (ApbCK116A, ApbCK121A, and ApbCS122A) (28Koonin E.V. J. Mol. Biol. 1993; 229: 1165-1174Crossref PubMed Scopus (259) Google Scholar); 2) conserved cysteine residues with spacing consistent with a metal-binding/thioredoxin motif (ApbCC283A and ApbCC286A) (45Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar); and 3) conserved serine (ApbCS182A) and nonconserved cysteine (ApbCC70A). 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Lewis J.A. Escalante-Semerena J.C. Downs D.M. J. Bacteriol. 2008; 190: 4596-4602Crossref PubMed Scopus (25) Google Scholar). Plasmids carrying alleles of apbC were introduced into two genetic backgrounds (apbC yggX and apbC purF) and provided the only source of apbC in the resulting strains. Each of the seven mutant alleles was tested for the ability to complement growth in both the low and high flux demand situations. The data in Table 2 show that substitutions in the Walker A (ApbCK116A, ApbCK121A, and ApbCS122A) and CXXC (ApbCC283A and ApbCC286A) motifs resulted in proteins unable to complement any of the three growth conditions. Changing the nonconserved Cys-70 to Ala had no detectable effect on in vivo function of the protein. In all cases, the empty vector and wild-type control plasmids displayed the growth patterns anticipated by past experiments (17Skovran E. Downs D.M. J. Bacteriol. 2003; 1
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