The metal binding site of zoocin A.
Direct metal analysis of the bacteriolytic exoenzyme zoocin A failed to unequivocally identify a putative metal cofactor; hence, indirect experiments utilizing NMR were undertaken to settle this question. Cd(2+) as a surrogate metal ion was reconstituted into EDTA-treated, metal-free recombinant zoocin, and (113)Cd-NMR was employed to explore binding in the protein for this ion. The Cd-substituted enzyme was found to have 80-85% of native streptococcolytic activity. A major (113)Cd resonance at 113.6 ppm was observed which with time split into resonances at 113.6 and 107.2 ppm. A minor (113)Cd resonance at 87.3 ppm was observed which increased in intensity with time. These Cd chemical shifts are indicative of two N atoms and two O atoms ligating directly to the metal site. On the basis of conserved amino acid residues in a homologous protein of known structure, LytM, the ligands in zoocin are tentatively assigned to H45, D49, H133, and some combination of water or buffer ions as the fourth oxygen donor in zoocin A. Comparison of the combined intensities for (113)Cd-substituted zoocin with a known quantity of another Cd-substituted protein gave Cd binding as approximately stoichiometric (1.2+/-0.2) with protein. Additional metal-removal and reconstitution experiments on the recombinant catalytic domain of zoocin implicate Zn(2+) as the metal cofactor. Therefore, the evidence supports zoocin as a single Zn(2+) ion binding metalloenzyme.
Chen
Yinghua
Y
Department of Chemistry, University of Alabama, Tuscaloosa, AL 35487-0336, USA.
Simmonds
Robin S
RS
Sloan
Gary L
GL
Timkovich
Russell
R
D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry*; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy / Q000379:methods / Q000592:standards; D009587:Nitrogen Isotopes; D011522:Protons; D012015:Reference Standards; D012680:Sensitivity and Specificity; D015032:Zinc / Q000737:chemistry
D001426:Bacterial Proteins; D007554:Isotopes; D009587:Nitrogen Isotopes; D011522:Protons; C106076:ZooA protein, Streptococcus equi; D002104:Cadmium; D015032:Zinc