PMID:2557837
| Citation |
Alwan, AF, Mgbeje, BI and Jordan, PM (1989) Purification and properties of uroporphyrinogen III synthase (co-synthase) from an overproducing recombinant strain of Escherichia coli K-12. Biochem. J. 264:397-402 |
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| Abstract |
The Escherichia coli hemD gene, encoding the enzyme uroporphyrinogen III synthase (co-synthase), was cloned into multi-copy plasmids in E. coli cells that were used to generate strains producing up to 1000 times the concentration of the synthase in the wild-type. The enzyme was purified to homogeneity from these strains in milligram amounts. The enzyme is a monomer of Mr 28,000 with an isoelectric point of 5.2 and a pH optimum of 7.8. The specific activity of the purified synthase is 1500 units/mg and the Km for the substrate, pre-uroporphyrinogen, is 5 microM. The N-terminal sequence of the enzyme is Ser-Ile-Leu-Val-Thr-Arg-Pro-Ser-Pro-Ala-Gly-, in agreement with the gene-derived protein sequence. The enzyme contains four 5,5'-dithiobis-(2-nitrobenzoic acid)-titratable groups, one reacting rapidly with the reagent and three further groups having lower reactivity. The enzyme is heat-sensitive, and during heat inactivation all four thiol groups become equally available for reaction. |
| Links | |
| Keywords |
Amino Acid Sequence; Chromatography; Chromatography, Gel; Chromatography, Ion Exchange; Cloning, Molecular; Dithionitrobenzoic Acid/pharmacology; Durapatite; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Genes, Bacterial; Hydro-Lyases/isolation & purification; Hydrogen-Ion Concentration; Hydroxyapatites; Kinetics; Molecular Sequence Data; Molecular Weight; Recombinant Proteins/isolation & purification; Uroporphyrinogen III Synthetase/genetics; Uroporphyrinogen III Synthetase/isolation & purification; Uroporphyrinogen III Synthetase/metabolism |
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