A Structural Basis for Half-of-the-sites Metal Binding Revealed in Drosophila melanogaster Porphobilinogen Synthase

Abstract

Porphobilinogen synthase (PBGS) proteins fall into several distinct groups with different metal ion requirements. Drosophila melanogaster porphobilinogen synthase (DmPBGS) is the first non-mammalian metazoan PBGS to be characterized. The sequence shows the determinants for two zinc binding sites known to be present in both mammalian and yeast PBGS, proteins that differ in the exhibition of half-of-the-sites metal binding. The pH-dependent activity of DmPBGS is uniquely affected by zinc. A tight binding catalytic zinc binds at 0.5/subunit with a K d well below M. A second inhibitory zinc exhibits a K d of ϳ5 M and appears to bind at a stoichiometry of 1/subunit. A molecular model of DmP-BGS suggests that the inhibitory zinc is located at a subunit interface using Cys-219 and His-10 as ligands. Zinc binding to this previously unknown inhibitory site is proposed to inhibit opening of the active site lid. As predicted, the DmPBGS mutant H10F is active but is not inhibited by zinc. H10F binds a catalytic zinc at 0.5/ subunit and binds a second nonessential and noninhibitory zinc at 0.5/subunit. This result reveals a structural basis for half-of-the-sites metal binding that is consistent with a reciprocating motion model for function of oligomeric PBGS. The porphobilinogen synthases (PBGS)1 are a family of highly homologous homo-octameric proteins responsible for catalyzing the first common step in the biosynthesis of a broad range of tetrapyrrole pigments such as heme, vitamin B12, chlorophyll, and cofactor F430 of the methanogenic bacteria (1). PBGS is also known as 5-aminolevulinate dehydratase or ALAD. The most significant phylogenetic difference among PBGS proteins is in the constellation of metal ions at catalytic and allosteric sites (2, 3). Yeast and mammalian PBGS share the sequence determinants for two zinc binding sites, one of which is absent from the PBGS of any archaea, bacteria, protist, or photosynthetic eucarya (3). The human and yeast PBGS both contain the sequence determinants for a catalytic active site zinc (also known as ZnB), as well as a second non-essential zinc (also known as ZnA) (4), but they differ in metal binding stoichiometry. For instance, in the case of human PBGS, the catalytic zinc shows half-of-the-sites binding at a stoichiometry of 4/homo-octamer (4) (PDB accession number 1E51), 2 whereas the fungal enzyme binds the catalytic zinc at 8/octamer (6) (PDB accession number 1AW5). Some bacterial PBGS show half-of-the-sites metal binding at catalytic and/or allosteric sites (e.g. Bradyrhizobium japonicum and Pseudomonas aeruginosa), whereas others (e.g. Escherichia coli) do not.The current sequence databases contain PBGS sequences from ϳ130 different organisms. Of these, the PBGS of Drosophila melanogaster (DmPBGS) is the only complete non-mammalian and non-fungal PBGS sequence that shows the sequence determinants for the two zinc binding sites found in yeast and mammalian PBGS. The gene encoding PBGS is absent from the completed genome of Caenorhabditis elegans and not yet verified in...

The Dance of Porphobilinogen Synthase in the Control of Tetrapyrrole Biosynthesis

“…Unlike the wild type protein, H10F is not inhibited by zinc in this concentration range. 74 Thus, the inhibitory zinc, which binds to His10, is also at a stoichiometry of 0.5 per subunit. Adapted from Ref.…”

“…Activity assays show inhibition associated with the looser binding zinc. 74 Panel B depicts a similar dialysis study for the D. melanogaster PBGS variant H10F. Triangles depict dialysis against various concentrations of zinc.…”

“…74 It has the sequence determinants for both the proximal and distal zinc binding sites, but the wild type protein binds zinc at a stoichiometry of 1.5 per subunit (12 per octamer), rather than the 1 per subunit that binds to mammalian PBGS. As seen in the mammalian protein, the proximal zinc binds very tightly (with a K d well below 1 µM) during turnover conditions at a stoichiometry of 0.5 per subunit.…”

“…Focus on the putative cysteine ligand to the distal zinc helped dissect this latter K d into two components that reflect zinc binding to Cys 119 in two different environments. Cys 119 74 Wild type protein also binds zinc at a stoichiometry of 1 per subunit. Activity assays show inhibition associated with the looser binding zinc.…”

Developmental profiles of 5-aminolevulinate, porphobilinogen and porphobilinogen synthase activity in Pieris brassicae related to the synthesis of the bilin-binding protein

“…It is surprising that despite the fundamental relevance of haem products, the enzymes of their biosynthetic pathway have been little investigated in insects to date, mostly in relation to the formation of mitochondrial cytochromes (e.g., Osanai and Rembold, 1974;Keeley, 1978a). Specifically with PBGS, more recent work backed by the progress in genomics was focused on the gene and protein structural levels of the Drosophila enzyme (Ruiz de Mena et al, 1999;Kundrat et al, 2003).…”