Probing the Active Site of Pseudomonas aeruginosa Porphobilinogen Synthase Using Newly Developed Inhibitors

Abstract

Porphobilinogen synthase catalyzes the first committed step of the tetrapyrrole biosynthesis pathway. In an aldol-like condensation, two molecules of 5-aminolevulinic acid (ALA) form the first pyrrole, porphobilinogen. Newly synthesized analogues of a reaction intermediate of porphobilinogen synthase have been employed in studying the active site and the catalytic mechanism of this early enzyme of tetrapyrrole biosynthesis. This study combines structural and kinetic evaluation of the inhibition potency of these inhibitors. In addition, one of the determined protein structures provides for the first time structural evidence of a magnesium ion in the active site. From these results, we can corroborate an earlier postulated enzymatic mechanism that starts with formation of a C-C bond, linking C3 of the A-side ALA to C4 of the P-side ALA through an aldole addition. The obtained data are discussed with respect to the current literature.

Structure of the Heme Biosynthetic Pseudomonas aeruginosa Porphobilinogen Synthase in Complex with the Antibiotic Alaremycin

“…3) (9, 14). Inhibitor binding is stabilized by two hydrogen bonds from the antibiotic carboxylate to Ser286, as described for 5-hydroxylevulinic acid, a substrate analogue, in the P site of P. aeruginosa PBGS (12). Alaremycin 2 thus mimics the P-site substrate ALA rather than the product PBG.…”

“…Clearly, the observed alare- (Table 3) would need to be further improved prior to any clinical applications. Previous investigations of the catalytic mechanism of P. aeruginosa PBGS by the use of synthetic inhibitors provide a rational basis for this (12). These inhibitors, with a (carbon) backbone of 11 atoms, bear ether, amine, thioether, sulfoxide, and sulfonyl groups at the central position, position 6.…”

“…During tetrapyrrole biosynthesis, four molecules of porphobilinogen are fused to generate the first tetrapyrrole, uroporphyrinogen III. Thereafter, the biosynthetic pathways of heme and chlorophyll separate from those of vitamin B 12 , siroheme, and factor F 430 due to distinct modifications of the initial tetrapyrrole skeleton (30).…”

“…The two ALA substrate molecules occupy two distinct binding sites in PBGS, referred to as the A and the P sites, to indicate that they contribute to porphobilinogen an acetic acid and a propanoic acid side chain, respectively. The ALA A and P sites are covalently bound through a stable Schiff base with lysine residues 205 and 260, respectively (12). Although PBGSs from different organisms differ with respect to the metal ion requirements and localization, the amino acid sequences are highly conserved between bacteria, archaea, and eukaryotes.…”

Synthesis of Bisubstrate Inhibitors of Porphobilinogen Synthase from Pseudomonas aeruginosa

“…Inhibition Studies. As an initial test, the IC 50 values of the bisubstrate analogs 7 -11 were determined (Table) according to the method described in [29]. When measuring the IC 50 values, the time-dependence of inhibition was detected.…”