Publications

• Bacterial heme biosynthesis and its biotechnological application.

  Frankenberg N., Moser J., Jahn D.

  Proteins carrying a prosthetic heme group are vital parts of bacterial energy conserving and stress response systems. They also mediate complex enzymatic reactions and regulatory processes. Here, we review the multistep biosynthetic pathway of heme formation including the enzymes involved and reac ... >> More

  Proteins carrying a prosthetic heme group are vital parts of bacterial energy conserving and stress response systems. They also mediate complex enzymatic reactions and regulatory processes. Here, we review the multistep biosynthetic pathway of heme formation including the enzymes involved and reaction mechanisms. Potential biotechnological implications are discussed. << Less

  Appl Microbiol Biotechnol 63:115-127(2003) [PubMed] [EuropePMC]

• Purification and properties of uroporphyrinogen III synthase from human erythrocytes.

  Tsai S.F., Bishop D.F., Desnick R.J.

  Uroporphyrinogen III synthase (hydroxymethylbilane hydro-lyase (cyclizing); EC 4.2.1.75), the fourth enzyme in the heme biosynthetic pathway, was purified to homogeneity from human erythrocytes. For enzyme purification and characterization, a sensitive coupled enzyme assay was used which generated ... >> More

  Uroporphyrinogen III synthase (hydroxymethylbilane hydro-lyase (cyclizing); EC 4.2.1.75), the fourth enzyme in the heme biosynthetic pathway, was purified to homogeneity from human erythrocytes. For enzyme purification and characterization, a sensitive coupled enzyme assay was used which generated the substrate, hydroxymethylbilane; the oxidized product, uroporphyrin III, was quantitated by high pressure liquid chromatography. Uroporphyrinogen III synthase was initially separated from delta-aminolevulinate dehydratase and hydroxymethylbilane synthase by a preparative anion exchange chromatographic step. Subsequent chromatography on hydroxyapatite, phenyl-Sepharose, and Sephadex G-100 purified the enzyme about 70,000-fold with an 8% yield. Homogeneous enzyme was obtained following a final C4-reversed phase high pressure liquid chromatographic step which removed a single major and several minor protein contaminants from the enzyme. The purified enzyme had a specific activity of over 300,000 units/mg, an isoelectric point of 5.5, and was thermolabile (t1/2 at 60 degrees C approximately 1 min). Molecular weight studies by gel filtration (Mr approximately equal to 30,000) and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately equal to 29,500) were consistent with the enzyme being a monomer. Using hydroxymethylbilane as substrate, the purified enzyme formed uroporphyrinogen III in the absence of hydroxymethylbilane synthase or other cofactors. The pH optimum was 7.4 and the Km for hydroxymethylbilane was 5-20 microM. The enzyme was activated by Na+, K+, Mg+, and Ca2+ and was inhibited by Cd2+, Cu2+, Hg2+, and Zn2+. Amino acid composition analysis was performed, and the N-terminal sequence, Met-Lys-Val-Leu-Leu-Leu, was determined by microsequencing. The availability of the purified enzyme should permit investigation of its reaction mechanism as well as facilitate biochemical and molecular studies of the genetic defect in congenital erythropoietic porphyria. << Less

  J Biol Chem 262:1268-1273(1987) [PubMed] [EuropePMC]

• Biosynthesis of the pigments of life: formation of the macrocycle.

  Battersby A.R., Fookes C.J., Matcham G.W., McDonald E.

  The organic nuclei of chlorophylls, haems, cytochromes and vitamin B12 are biosynthesised from a single tetrapyrrolic intermediate which has an unexpected, rearranged structure. The mechanism of biosynthesis of this key intermediate has now been characterised in detail. Some of the information the ... >> More

  The organic nuclei of chlorophylls, haems, cytochromes and vitamin B12 are biosynthesised from a single tetrapyrrolic intermediate which has an unexpected, rearranged structure. The mechanism of biosynthesis of this key intermediate has now been characterised in detail. Some of the information thereby obtained is also of use in the investigation of human diseases such as the porphyrias. << Less

  Nature 285:17-21(1980) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Crystal structure of human uroporphyrinogen III synthase.

  Mathews M.A., Schubert H.L., Whitby F.G., Alexander K.J., Schadick K., Bergonia H.A., Phillips J.D., Hill C.P.

  Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrino gen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F(430) and ... >> More

  Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrino gen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F(430) and vitamin B(12). U3S activity is essential in all organisms, and decreased activity in humans leads to the autosomal recessive disorder congenital erythropoetic porphyria. We have determined the crystal structure of recombinant human U3S at 1.85 A resolution. The protein folds into two alpha/beta domains connected by a beta-ladder. The active site appears to be located between the domains, and variations in relative domain positions observed between crystallographically independent molecules indicates the presence of flexibility that may be important in the catalytic cycle. Possible mechanisms of catalysis were probed by mutating each of the four invariant residues in the protein that have titratable side chains. Additionally, six other highly conserved and titratable side chains were also mutated. In no case, however, did one of these mutations abolish enzyme activity, suggesting that the mechanism does not require acid/base catalysis. << Less

  EMBO J. 20:5832-5839(2001) [PubMed] [EuropePMC]

• Structural diversity in metal ion chelation and the structure of uroporphyrinogen III synthase.

  Schubert H.L., Raux E., Matthews M.A., Phillips J.D., Wilson K.S., Hill C.P., Warren M.J.

  All tetrapyrroles are synthesized through a branched pathway, and although each tetrapyrrole receives unique modifications around the ring periphery, they all share the unifying feature of a central metal ion. Each pathway maintains a unique metal ion chelatase, and several tertiary structures hav ... >> More

  All tetrapyrroles are synthesized through a branched pathway, and although each tetrapyrrole receives unique modifications around the ring periphery, they all share the unifying feature of a central metal ion. Each pathway maintains a unique metal ion chelatase, and several tertiary structures have been determined, including those of the protoporphyrin ferrochelatase from both human and Bacillus subtilus, and the cobalt chelatase CbiK. These enzymes exhibit strong structural similarity and appear to function by a similar mechanism. Met8p, from Saccharomyces cerevisiae, catalyses ferrochelation during the synthesis of sirohaem, and the structure reveals a novel chelatase architecture whereby both ferrochelation and NAD(+)-dependent dehydrogenation take place in a single bifunctional active site. Asp-141 appears to participate in both catalytic reactions. The final common biosynthetic step in tetrapyrrole biosynthesis is the generation of uroporphyrinogen by uroporphyrinogen III synthase, whereby the D ring of hydroxymethylbilane is flipped during ring closure to generate the asymmetrical structure of uroporphyrinogen III. The recently derived structure of uroporphyrinogen III synthase reveals a bi-lobed structure in which the active site lies between the domains. << Less

  Biochem Soc Trans 30:595-600(2002) [PubMed] [EuropePMC]