Pseudomonas aeruginosa Contains a Novel Type V Porphobilinogen Synthase with No Required Catalytic Metal Ions

Porphobilinogen synthases (PBGS) are metalloenzymes that catalyze the first common step in tetrapyrrole biosynthesis. The PBGS enzymes have previously been categorized into four types (I−IV) by the number of Zn2+ and/or Mg2+ utilized at three different metal binding sites termed A, B, and C. In this study Pseudomonas aeruginosa PBGS is found to bind only four Mg2+ per octamer as determined by atomic absorption spectroscopy, in the presence or absence of substrate/product. This is the lowest number of bound metal ions yet found for PBGS where other enzymes bind 8−16 divalent ions. These four Mg2+ allosterically stimulate a metal ion independent catalytic activity, in a fashion dependent upon both pH and K+. The allosteric Mg2+ of PBGS is located in metal binding site C, which is outside the active site. No evidence is found for metal binding to the potential high-affinity active site metal binding sites A and/or B. P. aeruginosa PBGS was investigated using Mn2+ as an EPR probe for Mg2+, and the active site was investigated using [3,5-13C]porphobilinogen as an NMR probe. The magnetic resonance data exclude the direct involvement of Mg2+ in substrate binding and product formation. The combined data suggest that P. aeruginosa PBGS represents a new type V enzyme. Type V PBGS has the remarkable ability to synthesize porphobilinogen in a metal ion independent fashion. The total metal ion stoichiometry of only 4 per octamer suggests half-sites reactivity.