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 Zn(2+) and/or Mg(2+) utilized at three different metal binding sites termed A, B, and C. In this study Pseudomonas aeruginosa PBGS is found to bind only four Mg(2+) 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 Mg(2+) allosterically stimulate a metal ion independent catalytic activity, in a fashion dependent upon both pH and K(+). The allosteric Mg(2+) 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 Mn(2+) as an EPR probe for Mg(2+), and the active site was investigated using [3,5-(13)C]porphobilinogen as an NMR probe. The magnetic resonance data exclude the direct involvement of Mg(2+) 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.
Frankenberg
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N
Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, Albertstrasse 21, 79104 Freiburg im Breisgau, Germany.
Jahn
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D
Jaffe
E K
EK
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001665:Binding Sites; D002247:Carbon Isotopes; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D000623:Porphobilinogen Synthase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013054:Spectrophotometry, Atomic
D002247:Carbon Isotopes; D008670:Metals; D008345:Manganese; D000623:Porphobilinogen Synthase; D008274:Magnesium