The synthesis of tetrapyrroles is essential to all phyla. Porphobilinogen synthase (PBGS) is a zinc metalloenzyme that catalyzes the formation of porphobilinogen, the monopyrrole precursor of all biological tetrapyrroles. The enzyme from various organisms shows considerable sequence conservation, suggesting a common fold, quaternary structure, and catalytic mechanism. Escherichia coli and plant PBGS are activated by magnesium, a property that is absent from mammalian PBGS. This stimulatory Mg(II) is called Mgc. Mgc is not required for activity and is distinct from the two zinc ions (ZnA and ZnB) common to mammalian and E. coli PBGS (PBGSE.coli). For PBGSE.coli, both the Km for the substrate 5-aminolevulinic acid (ALA) and the Vmax are altered by the presence of Mgc; Mg(II) causes the Km to drop from approximately 3 to 0.30 mM and the maximum specific activity to increase from 23 to 50 mumol h-1 mg-1. Mgc also causes the saturating concentration of the required Zn(II) to decrease from 0.1 mM to 10 microM. Maximal activation by Mg(II) occurs at 0.5 mM; thus, in E. coli the Mgc site is probably saturated under physiological conditions. Mn(II) is a good substitute for Mgc, giving a comparable increase in catalytic activity. Consequently, Mn(II) has been used as an EPR active probe of the Mgc binding site. Mn(II) binds at a stoichiometry of eight ions per enzyme octamer. The X- and Q-band EPR spectra reflect a single type of binding site with rhombic symmetry and are consistent with oxygen and/or nitrogen ligands. The addition of unlabeled or 1-13C-labeled ALA does not significantly affect the Mn(II) EPR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)

Porphobilinogen synthase (PBGS) is present in all organisms that synthesize tetrapyrroles such as heme, chlorophyll, and vitamin B(12). The homooctameric metalloenzyme catalyzes the condensation of two 5-aminolevulinic acid molecules to form the tetrapyrrole precursor porphobilinogen. An artificial gene encoding PBGS of pea (Pisum sativum L.) was designed to overcome previous problems during bacterial expression caused by suboptimal codon usage and was constructed by recursive polymerase chain reaction from synthetic oligonucleotides. The recombinant 330 residue enzyme without a putative chloroplast transit peptide was expressed in Escherichia coli and purified in 100-mg quantities. The specific activity is protein concentration dependent, which indicates that a maximally active octamer can dissociate into less active smaller units. The enzyme is most active at slightly alkaline pH; it shows two pK(a) values of 7.4 and 9.7. Atomic absorption spectroscopy shows maximal binding of three Mg(II) per subunit; kinetic data support two functionally distinct types of Mg(II) and the third appears to be nonphysiologic and inhibitory. Analysis of the protein concentration dependence of the specific activity suggests that the minimal functional unit is a tetramer. A model of octameric pea PBGS was built to predict the location of intermolecular disulfide linkages that were revealed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As verified by site-specific mutagenesis, disulfide linkages can form between four cysteines per octamer, each located five amino acids from the C-terminus. These data are consistent with the protein undergoing conformational changes and the idea that whole-body motion can occur between subunits.

Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B 12 ). Two common alleles encoding human PBGS, K59 and N59, have been correlated with differential susceptibility of humans to lead poisoning. However, a model for human PBGS based on homologous crystal structures shows the location of the allelic variation to be distant from the active site with its two Zn(II). Previous microbial expression systems for human PBGS have resulted in a poor yield. Here, an artificial gene encoding human PBGS was constructed by recursive polymerase chain reaction from synthetic oligonucleotides to rectify this problem. The artificial gene was made to resemble the highly expressed homologous Escherichia coli hemB gene and to remove rare codons that can confound heterologous protein expression in E. coli. We have expressed and purified recombinant human PBGS variants K59 and N59 in 100-mg quantities. Both human PBGS proteins purified with eight Zn(II)/octamer; Zn(II) binding was shown to be pH-dependent; and Pb(II) could displace some of the Zn(II). However, there was no differential displacement of Zn(II) by Pb(II) between K59 and N59, and simple Pb(II) inhibition studies revealed no allelic difference.

5-Chloro[1,4-13C]levulinic acid ([1,4-13C]CLA) is an active site-directed inactivator of porphobilinogen synthase (PBGS). PBGS asymmetrically condenses two molecules of 5-aminolevulinic acid (ALA) which are called A-side ALA and P-side ALA in reference to their fates as the acetyl and propionyl halves of the product. [1,4-13C]CLA modifies bovine PBGS at the A-side ALA binding site. The C4 chemical shift indicates an intact keto moiety; the C1 chemical shift indicates a deprotonated carboxyl group. In contrast, [1,4-13C]CLA modification of Escherichia coli PBGS is heterogeneous and occurs preferentially at the P-side ALA binding site. The C1 chemical shifts indicate substantially deprotonated carboxylic acid groups. For one of four observed forms of [1,4-13C]CLA-modified E. coli PBGS, an analog of the P-site Schiff base is found. Bovine and E. coli PBGS contain two different zincs, ZnA and ZnB. Past results placed ZnA near A-side ALA. [1,4-13C]CLA modifies E. coli PBGS at Cys119 or Cys129, which is part of a four-cysteine cluster implicated in binding ZnB. This result places ZnB near P-side ALA. E. coli PBGS binds a third type of divalent metal, MgC or MnC, which is found to have no significant effect on the 13C NMR spectrum of the [1,4-13C]CLA-modified protein.

Ageing is characterized by a decline in stem cell functionality leading to dampened tissue regeneration. While the expression of microRNAs across multiple species is markedly altered with age, the mechanism by which they govern stem cell-sustained tissue regeneration is unknown. We report that in the Drosophila testis, the conserved miR-9a is expressed in germline stem cells and its levels are significantly elevated during ageing. Transcriptome and functional analyses show that miR-9a directly regulates the expression of the adhesion molecule N-cadherin (N-cad). miR-9a null mutants maintain a higher number of stem cells even in the aged tissue. Remarkably, this rise fails to improve tissue regeneration and results in reduced male fertility. Similarly, overexpression of N-cad also results in elevated stem cell number and decreased regeneration. We propose that miR-9a downregulates N-cad to enable adequate detachment of stem cells toward differentiation, thus providing the necessary directionality toward terminal differentiation and spermatogenesis.