Isolated Bacillus subtilis HemY has coproporphyrinogen III to coproporphyrin III oxidase activity.
Oxidation of coproporphyrinogen III to coproporphyrin III is found in extracts of Escherichia coli cells containing the Bacillus subtilis HemY protein (M. Hansson and L. Hederstedt, J. Bacteriol. 176, 5962-5970). We have analysed whether this activity is due to the heterologous expression system, since it in vivo would lead to disruption of the heme biosynthetic pathway. B. subtilis hemY was fused in its 3'-end to a polynucleotide encoding six histidine residues and expressed from plasmids in both E. coli and B. subtilis. The His6-tagged HemY protein extracted from membranes using non-ionic detergent was purified by Ni2+ affinity chromatography. Isolated HemY fusion protein synthesised in E. coli and B. subtilis oxidised coproporphyrinogen III to coproporphyrin III. No direct formation of protoporphyrin IX from coproporphyrinogen III could be detected. Our results suggest that the coproporphyrinogen III to coproporphyrin III activity of HemY is either avoided in B. subtilis in vivo or that coproporphyrin III is a heme biosynthetic intermediate in this bacterium.
Hansson
M
M
Carlsberg Laboratory, Department of Physiology, Copenhagen Valby, Denmark. hansson@biobase.dk
Gustafsson
M C
MC
Kannangara
C G
CG
Hederstedt
L
L
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D003305:Coproporphyrinogens / Q000378:metabolism*; D003306:Coproporphyrins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006639:Histidine; D008969:Molecular Sequence Data; D009532:Nickel; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase; D011993:Recombinant Fusion Proteins; D012995:Solubility
D003305:Coproporphyrinogens; D003306:Coproporphyrins; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; C026971:coproporphyrin III; C028026:coproporphyrinogen III; D006639:Histidine; D009532:Nickel; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050768:Protoporphyrinogen Oxidase