The CoxD protein, a novel AAA+ ATPase involved in metal cluster assembly: hydrolysis of nucleotide-triphosphates and oligomerization

• Published in: PloS one Vol. 7; no. 10; p. e47424
• Main Authors: Maisel, Tobias, Joseph, Stephanie, Mielke, Thorsten, Bürger, Jörg, Schwarzinger, Stephan, Meyer, Ortwin
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 15.10.2012; Public Library of Science (PLoS)
• Subjects: Adenosine Diphosphate - metabolism; Adenosine triphosphatase; Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - metabolism; Adenosine triphosphate; Adenosine Triphosphate - metabolism; Aldehyde Oxidoreductases - metabolism; Amino acids; Analysis; Assembly; ATP; Bacteria; Bacterial corrosion; Bacterial genetics; Biology; Biophysics; Biosynthesis; Catalytic Domain; Centrifugation; Chemistry; Circular dichroism; Copper; Density gradients; Dichroism; Dichroism (Pleochroism); Dilution; Dimers; E coli; Electron microscopy; Enzymes; Escherichia coli; Gel filtration; Genes; Genetic aspects; Guanosine triphosphatases; Health physics; Hexamers; Hydrolysis; Inclusion bodies; Inserts; Kinases; Magnesium; Membrane proteins; Metal clusters; Metals - chemistry; Metals - metabolism; Multienzyme Complexes - metabolism; Nucleotides - chemistry; Nucleotides - metabolism; Oligomerization; Oxidoreductases; Phosphates; Protein Conformation; Protein Structure, Secondary; Proteins; Proteobacteria - enzymology; Rhodobacter sphaeroides; Spectroscopy; Structural members; Sucrose; Sugar; Sulfur; Synechocystis; Three dimensional models; Transmission electron microscopy; Urea; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0047424

CoxD of the α-proteobacterium Oligotropha carboxidovorans is a membrane protein which is involved in the posttranslational biosynthesis of the [CuSMoO₂] cluster in the active site of the enzyme CO dehydrogenase. The bacteria synthesize CoxD only in the presence of CO. Recombinant CoxD produced in E. coli K38 pGP1-2/pETMW2 appeared in inclusion bodies from where it was solubilized by urea and refolded by stepwise dilution. Circular dichroism spectroscopy revealed the presence of secondary structural elements in refolded CoxD. CoxD is a P-loop ATPase of the AAA-protein family. Refolded CoxD catalyzed the hydrolysis of MgATP yielding MgADP and inorganic phosphate at a 1∶1∶1 molar ratio. The reaction was inhibited by the slow hydrolysable MgATP-γ-S. GTPase activity of CoxD did not exceed 2% of the ATPase activity. Employing different methods (non linear regression, Hanes and Woolf, Lineweaver-Burk), preparations of CoxD revealed a mean K(M) value of 0.69±0.14 mM ATP and an apparent V(max) value of 19.3±2.3 nmol ATP hydrolyzed min⁻¹ mg⁻¹. Sucrose density gradient centrifugation and gel filtration showed that refolded CoxD can exist in various multimeric states (2-mer, 4-mer or 6-mer), preferentially as hexamer or dimer. Within weeks the hexamer dissociates into the dimer, a process which can be reversed by MgATP or MgATP-γ-S within hours. Only the hexamers and the dimers exhibited MgATPase activity. Transmission electron microscopy of negatively stained CoxD preparations revealed distinct particles within a size range of 10-16 nm, which further corroborates the oligomeric organization. The 3D structure of CoxD was modeled with the 3D structure of BchI from Rhodobacter capsulatus as template. It has the key elements of an AAA+ domain in the same arrangement and at same positions as in BchI and displays the characteristic inserts of the PS-II-insert clade. Possible functions of CoxD in [CuSMoO₂] cluster assembly are discussed.