title: Fine-tuning of Substrate Affinity Leads to Alternative Roles of Mycobacterium tuberculosis Fe2+-ATPases.
abstract: Little is known about iron efflux transporters within bacterial systems. Recently, the participation of Bacillus subtilis PfeT, a P1B4-ATPase, in cytoplasmic Fe(2+) efflux has been proposed. We report here the distinct roles of mycobacterial P1B4-ATPases in the homeostasis of Co(2+) and Fe(2+) Mutation of Mycobacterium smegmatis ctpJ affects the homeostasis of both ions. Alternatively, an M. tuberculosis ctpJ mutant is more sensitive to Co(2+) than Fe(2+), whereas mutation of the homologous M. tuberculosis ctpD leads to Fe(2+) sensitivity but no alterations in Co(2+) homeostasis. In vitro, the three enzymes are activated by both Fe(2+) and Co(2+) and bind 1 eq of either ion at their transport site. However, equilibrium binding affinities and activity kinetics show that M. tuberculosis CtpD has higher affinity for Fe(2+) and twice the Fe(2+)-stimulated activity than the CtpJs. These parameters are paralleled by a lower activation and affinity for Co(2+) Analysis of Fe(2+) and Co(2+) binding to CtpD by x-ray absorption spectroscopy shows that both ions are five- to six-coordinate, constrained within oxygen/nitrogen environments with similar geometries. Mutagenesis studies suggest the involvement of invariant Ser, His, and Glu residues in metal coordination. Interestingly, replacement of the conserved Cys at the metal binding pocket leads to a large reduction in Fe(2+) but not Co(2+) binding affinity. We propose that CtpJ ATPases participate in the control of steady state Fe(2+) levels. CtpD, required for M. tuberculosis virulence, is a high affinity Fe(2+) transporter involved in the rapid response to iron dyshomeostasis generated upon redox stress.
authors.affiliation: From the Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609.
authors.forename: Sarju J
authors.initials: SJ
authors.lastname: Patel
authors.affiliation: Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan 48201.
authors.forename: Brianne E
authors.initials: BE
authors.lastname: Lewis
authors.affiliation: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655, and.
authors.forename: Jarukit E
authors.initials: JE
authors.lastname: Long
authors.affiliation: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655, and.
authors.forename: Subhalaxmi
authors.initials: S
authors.lastname: Nambi
authors.affiliation: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655, and Howard Hughes Medical Institute, Chevy Chase, Maryland 20815.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Sassetti
authors.affiliation: Department of Pharmaceutical Sciences, Wayne State University, Detroit, Michigan 48201.
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Stemmler
authors.affiliation: From the Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609, arguello@wpi.edu.
authors.forename: José M
authors.initials: JM
authors.lastname: Argüello
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D013379:Substrate Specificity; D014376:Tuberculosis / Q000378:metabolism / Q000382:microbiology; D014774:Virulence; D056928:X-Ray Absorption Spectroscopy
chemical_list: D001426:Bacterial Proteins; D008670:Metals; D007501:Iron; D000251:Adenosine Triphosphatases
keywords: ATPase; Mycobacterium tuberculosis; P1B4-ATPase; iron; metal homeostasis; metal ion-protein interaction; transport metal