Purification and properties of uroporphyrinogen III synthase (co-synthase) from an overproducing recombinant strain of Escherichia coli K-12.
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.
Alwan
A F
AF
Department of Biochemistry, University of Southampton, U.K.
Mgbeje
B I
BI
Jordan
P M
PM
D000595:Amino Acid Sequence; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification; D014576:Uroporphyrinogen III Synthetase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D006882:Hydroxyapatites; D011994:Recombinant Proteins; D017886:Durapatite; D004228:Dithionitrobenzoic Acid; D006836:Hydro-Lyases; D014576:Uroporphyrinogen III Synthetase