The metal binding site of zoocin A

Abstract

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 Cdsubstituted enzyme was found to have 75% 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 nitrogen atoms and two oxygen atoms ligating directly to the metal site. Based upon 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 Cdbinding 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 ion binding metalloenzyme.

Solution structure of the Cys74 to Ala74 mutant of the recombinant catalytic domain of Zoocin A

“…This strategy is akin to incorporating a pre‐ordained planar heme ring into a hemoprotein NMR structure calculation. Since the fourth ligand of tetrahedral Zn 2+ could also not be identified, a single oxygen atom was incorporated wholly consistent with the crystal structures and a previous atom‐type identification by NMR …”

“…Since the fourth ligand of tetrahedral Zn 21 could also not be identified, a single oxygen atom was incorporated wholly consistent with the crystal structures and a previous atom-type identification by NMR. 2 Ambiguous NOE assignments were refined based on the current structural model, and then hydrogen bond restraints and chemical shift restraints were added. Thirteen proton chemical shifts affected by aromatic ring currents were identified in the later stages of refinement; the distances between these protons and the ring centers were set to 2.5-4 Å .…”

“…BLAST sequence homology analysis showed that CAT of zoocin A has homology to the M23 family of peptidases, including the catalytic domain of Staphylococcus simulans lysostaphin (41% sequence identity) and the catalytic domain of Staphylococcus aureus LytM (35% sequence identity). Both lysostaphin and LytM are M23 endopeptidases using Zn 2+ ion as the metal cofactor . The single Zn 2+ ion in M23 peptidases is coordinated by two conserved motifs HXXXD and HXH (where X is any amino acid).…”

“…Both lysostaphin and LytM are M23 endopeptidases using Zn 21 ion as the metal cofactor. 2 The single Zn 21 ion in M23 peptidases is coordinated by two conserved motifs HXXXD and HXH (where X is any amino acid). Previous NMR studies demonstrated that zoocin A also consists of one Zn 21 ion tetrahedrally coordinated by His45 and Asp49 in the HXXXD motif, His133 in the HXH motif, and a fourth oxygen from water or buffer ions.…”

“…Previous NMR studies demonstrated that zoocin A also consists of one Zn 21 ion tetrahedrally coordinated by His45 and Asp49 in the HXXXD motif, His133 in the HXH motif, and a fourth oxygen from water or buffer ions. 2 In the standard dye release assay from purified, insoluble peptidoglycan fragments from the cell wall of susceptible bacteria, 3 the site specific mutant rCAT C74A had 92% 6 9% of the enzymatic activity of wild type. The mutation prevented the formation of a homodimer through a disulfide bond, making it easier to manipulate the protein in aerated solutions without protecting reagents.…”