Publications

• Rhodobacter capsulatus porphobilinogen synthase, a high activity metal ion independent hexamer.

  Bollivar D.W., Clauson C., Lighthall R., Forbes S., Kokona B., Fairman R., Kundrat L., Jaffe E.K.

  <h4>Background</h4>The enzyme porphobilinogen synthase (PBGS), which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion ... >> More

  <h4>Background</h4>The enzyme porphobilinogen synthase (PBGS), which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage. This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium Rhodobacter capsulatus.<h4>Results</h4>The gene for R. capsulatus PBGS was amplified from genomic DNA and sequencing revealed errors in the sequence database. R. capsulatus PBGS was heterologously expressed in E. coli and purified to homogeneity. Analysis of an unusual phylogenetic variation in metal ion usage by PBGS enzymes predicts that R. capsulatus PBGS does not utilize metal ions such as Zn2+, or Mg2+, which have been shown to act in other PBGS at either catalytic or allosteric sites. Studies with these ions and chelators confirm the predictions. A broad pH optimum was determined to be independent of monovalent cations, approximately 8.5, and the Km value shows an acidic pKa of approximately 6. Because the metal ions of other PBGS affect the quaternary structure, gel permeation chromatography and analytical ultracentrifugation experiments were performed to examine the quaternary structure of metal ion independent R. capsulatus PBGS. The enzyme was found to be predominantly hexameric, in contrast with most other PBGS, which are octameric. A protein concentration dependence to the specific activity suggests that the hexameric R. capsulatus PBGS is very active and can dissociate to smaller, less active, species. A homology model of hexameric R. capsulatus PBGS is presented and discussed.<h4>Conclusion</h4>The evidence presented in this paper supports the unusual position of the R. capsulatus PBGS as not requiring any metal ions for function. Unlike other wild-type PBGS, the R. capsulatus protein is a hexamer with an unusually high specific activity when compared to other octameric PBGS proteins. << Less

  BMC Biochem. 5:17-17(2004) [PubMed] [EuropePMC]

• Bradyrhizobium japonicum delta-aminolevulinic acid dehydratase is essential for symbiosis with soybean and contains a novel metal-binding domain.

  Chauhan S., O'Brian M.R.

  The Bradyrhizobium japonicum hemA gene product delta-aminolevulinic acid (ALA) synthase is not required for symbiosis of that bacterium with soybean. Hence, the essentiality of the subsequent heme synthesis enzyme, ALA dehydratase, was examined. The B. japonicum ALA dehydratase gene, termed hemB, ... >> More

  The Bradyrhizobium japonicum hemA gene product delta-aminolevulinic acid (ALA) synthase is not required for symbiosis of that bacterium with soybean. Hence, the essentiality of the subsequent heme synthesis enzyme, ALA dehydratase, was examined. The B. japonicum ALA dehydratase gene, termed hemB, was isolated and identified on the basis of its ability to confer hemin prototrophy and enzyme activity on an Escherichia coli hemB mutant, and it encoded a protein that was highly homologous to ALA dehydratases from diverse organisms. A novel metal-binding domain in the B. japonicum ALA dehydratase was identified that is a structural composite of the Mg(2+)-binding domain found in plant ALA dehydratases and the Zn(2+)-binding region of nonplant ALA dehydratases. Enzyme activity in dialyzed extracts of cells that overexpressed the hemB gene was reconstituted by the addition of Mg2+ but not by addition of Zn2+, indicating that the B. japonicum ALA dehydratase is similar to the plant enzymes with respect to its metal requirement. Unlike the B. japonicum hemA mutant, the hemB mutant strain KP32 elicited undeveloped nodules on soybean, indicated by the lack of nitrogen fixation activity and plant hemoglobin. We conclude that the hemB gene is required for nodule development and propose that B. japonicum ALA dehydratase is the first essential bacterial enzyme for B. japonicum heme synthesis in soybean root nodules. In addition, we postulate that ALA is the only heme intermediate that can be translocated from the plant to the endosymbiont to support bacterial heme synthesis in nodules. << Less

  J. Bacteriol. 175:7222-7227(1993) [PubMed] [EuropePMC]

• Allostery and the dynamic oligomerization of porphobilinogen synthase.

  Jaffe E.K., Lawrence S.H.

  The structural basis for allosteric regulation of porphobilinogen synthase (PBGS) is modulation of a quaternary structure equilibrium between octamer and hexamer (via dimers), which is represented schematically as 8mer ⇔ 2mer ⇔ 2mer∗⇔ 6mer∗. The "∗" represents a reorientation between two domains o ... >> More

  The structural basis for allosteric regulation of porphobilinogen synthase (PBGS) is modulation of a quaternary structure equilibrium between octamer and hexamer (via dimers), which is represented schematically as 8mer ⇔ 2mer ⇔ 2mer∗⇔ 6mer∗. The "∗" represents a reorientation between two domains of each subunit that occurs in the dissociated state because it is sterically forbidden in the larger multimers. Allosteric effectors of PBGS are both intrinsic and extrinsic and are phylogenetically variable. In some species this equilibrium is modulated intrinsically by magnesium which binds at a site specific to the 8mer. In other species this equilibrium is modulated intrinsically by pH with the guanidinium group of an arginine being spatially equivalent to the allosteric magnesium ion. In humans, disease associated variants all shift the equilibrium toward the 6mer∗ relative to wild type. The 6mer∗ has a surface cavity that is not present in the 8mer and is proposed as a small molecule allosteric binding site. In silico and in vitro approaches have revealed species-specific allosteric PBGS inhibitors that stabilize the 6mer∗. Some of these inhibitors are drugs in clinical use leading to the hypothesis that extrinsic allosteric inhibition of human PBGS could be a mechanism for drug side effects. << Less

  Arch Biochem Biophys 519:144-153(2012) [PubMed] [EuropePMC]

• The molecular mechanism of lead inhibition of human porphobilinogen synthase.

  Jaffe E.K., Martins J., Li J., Kervinen J., Dunbrack R.L. Jr.

  Human porphobilinogen synthase (PBGS) is a main target in lead poisoning. Human PBGS purifies with eight Zn(II) per homo-octamer; four ZnA have predominantly nonsulfur ligands, and four ZnB have predominantly sulfur ligands. Only four Zn(II) are required for activity. To better elucidate the roles ... >> More

  Human porphobilinogen synthase (PBGS) is a main target in lead poisoning. Human PBGS purifies with eight Zn(II) per homo-octamer; four ZnA have predominantly nonsulfur ligands, and four ZnB have predominantly sulfur ligands. Only four Zn(II) are required for activity. To better elucidate the roles of Zn(II) and Pb(II), we produced human PBGS mutants that are designed to lack either the ZnA or ZnB sites. These proteins, MinusZnA (H131A, C223A) and MinusZnB (C122A, C124A, C132A), each become purified with four Zn(II) per octamer, thus confirming an asymmetry in the human PBGS structure. MinusZnA is fully active, whereas MinusZnB is far less active, verifying an important catalytic role for ZnB and the removed cysteine residues. Kinetic properties of the mutants and wild type proteins are described. Comparison of Pb(II) inhibition of the mutants shows that ligands to both ZnA and ZnB interact with Pb(II). The ZnB ligands preferentially interact with Pb(II). At least one ZnA ligand is responsible for the slow tight binding behavior of Pb(II). The data support a novel model where a high affinity lead site is a hybrid of the ZnA and ZnB sites. We propose that the lone electron pair of Pb(II) precludes Pb(II) to function in PBGS catalysis. << Less

  J. Biol. Chem. 276:1531-1537(2001) [PubMed] [EuropePMC]

• Lead poisoning, haem synthesis and 5-aminolaevulinic acid dehydratase.

  Warren M.J., Cooper J.B., Wood S.P., Shoolingin-Jordan P.M.

  In mammals and yeast, 5-aminolaevulinic acid dehydratase is a zinc-dependent enzyme that catalyses the synthesis of porphobilinogen-the pyrrole building block that is incorporated into all modified tetrapyrroles, including haem, chlorophyll and vitamin B12. The X-ray structure of this enzyme revea ... >> More

  In mammals and yeast, 5-aminolaevulinic acid dehydratase is a zinc-dependent enzyme that catalyses the synthesis of porphobilinogen-the pyrrole building block that is incorporated into all modified tetrapyrroles, including haem, chlorophyll and vitamin B12. The X-ray structure of this enzyme reveals how substitution of the catalytically important zinc ion by lead inactivates the enzyme and causes a form of pseudo-porphyria. << Less

  Trends Biochem Sci 23:217-221(1998) [PubMed] [EuropePMC]

• Porphobilinogen synthase from pea: expression from an artificial gene, kinetic characterization, and novel implications for subunit interactions.

  Kervinen J., Dunbrack R.L. Jr., Litwin S., Martins J., Scarrow R.C., Volin M., Yeung A.T., Yoon E., Jaffe E.K.

  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 artificia ... >> More

  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. << Less

  Biochemistry 39:9018-9029(2000) [PubMed] [EuropePMC]

• Probing the active site of Pseudomonas aeruginosa porphobilinogen synthase using newly developed inhibitors.

  Frere F., Nentwich M., Gacond S., Heinz D.W., Neier R., Frankenberg-Dinkel N.

  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 synth ... >> More

  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. << Less

  Biochemistry 45:8243-8253(2006) [PubMed] [EuropePMC]

• Catalytic mechanism of porphobilinogen synthase: the chemical step revisited by QM/MM calculations.

  Tian B.X., Erdtman E., Eriksson L.A.

  Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation and cyclization of two 5-aminolevulinic acid (5-ALA) substrate molecules to give porphobilinogen (PBG). The chemical step of PBGS is herein revisited using QM/MM (ONIOM) calculations. Two different protonation states and several ... >> More

  Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation and cyclization of two 5-aminolevulinic acid (5-ALA) substrate molecules to give porphobilinogen (PBG). The chemical step of PBGS is herein revisited using QM/MM (ONIOM) calculations. Two different protonation states and several different mechanisms are considered. Previous mechanisms based on DFT-only calculations are shown unlikely to occur. According to these new calculations, the deprotonation step rather than ring closure is rate-limiting. Both the C-C bond formation first mechanism and the C-N bond formation first mechanism are possible, depending on how the A-site ALA binds to the enzyme. We furthermore propose that future work should focus on the substrate binding step rather than the enzymatic mechanism. << Less

  J Phys Chem B 116:12105-12112(2012) [PubMed] [EuropePMC]

• Characterization of the role of the stimulatory magnesium of Escherichia coli porphobilinogen synthase.

  Jaffe E.K., Ali S., Mitchell L.W., Taylor K.M., Volin M., Markham G.D.

  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, ... >> More

  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) << Less

  Biochemistry 34:244-251(1995) [PubMed] [EuropePMC]