Expression of a cloned protoporphyrinogen oxidase.
The previously cloned hem Y gene of Bacillus subtilis (Hansson, M., and Hederstedt, L. (1992) J. Bacteriol. 174, 8081-8093) has been expressed in Escherichia coli. The expressed protein has been shown to be the penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4) and, thus, the gene designation should be hem G. This represents the first report of the expression of a cloned protoporphyrinogen oxidase from any source. The enzyme is present in the soluble cytoplasmic fraction and is, thus, unlike all previously reported eukaryotic or prokaryotic protoporphyrinogen oxidases, which are membrane-bound. It utilizes molecular oxygen as a terminal electron acceptor, and protoporphyrinogen IX, mesoporphyrinogen IX, and coproporphyrinogen III serve as substrates. The diphenyl ether herbicide acifluorfen, which is a strong inhibitor of the eukaryotic enzyme, is only weakly inhibitory. The enzyme has a predicted molecular weight of 51,200, which corresponds well with molecular weight determination via high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis. In addition the enzyme contains a putative dinucleotide binding region at the amino terminus, which is consistent with the previously demonstrated presence of a flavin moiety in the characterized mammalian enzymes.
Dailey
T A
TA
Department of Microbiology, University of Georgia, Athens 30602-2605.
Meissner
P
P
Dailey
H A
HA
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000235:genetics*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D013347:Subcellular Fractions / Q000201:enzymology
D001426:Bacterial Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase