Mouse coproporphyrinogen oxidase is a copper-containing enzyme: expression in Escherichia coli and site-directed mutagenesis.
We previously isolated cDNA for mouse coproporphyrinogen oxidase (CPO) and provided evidence for the induction of mRNA during differentiation of murine erythroleukemia cells (Kohno et al. (1993) J. Biol. Chem. 268, 21359-21363). To better understand the structure and the mechanisms of reaction of the enzyme, we expressed mouse CPO in Escherichia. coli and purified it to a homogeneity. Analysis of the metal content revealed that the recombinant mouse CPO contains one copper atom per polypeptide chain. When the bacterial cells were treated with D-penicillamine, a copper chelator, formation of the active CPO was partially reduced. Addition of Cu2+ in minimal medium resulted in 6-fold higher level of CPO activity. These results suggest that expression of active mouse CPO in E. coli depended on the presence of Cu2+ in the culture medium. To elucidate the apparent involvement of Cu2+ in enzyme function, a series of mutant enzymes, whose highly conserved histidine and cysteine residues were individually converted to alanine residue, were prepared by site-directed mutagenesis. Mutant enzymes were expressed in E. coli and their activities examined. Mutation at histidine 158 resulted in a complete loss of enzyme activity, yet the enzyme protein was expressed at a comparable level. Concomitantly, only a trace amount of Cu2+ was detected in the purified H158A enzyme. We propose that mouse CPO is copper-containing enzyme and Cu2+ interacts with a conserved histidine residue.
Kohno
H
H
Department of Hygiene, Kansai Medical University, Osaka, Japan.
Furukawa
T
T
Tokunaga
R
R
Taketani
S
S
Yoshinaga
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002614:Chelating Agents / Q000494:pharmacology; D003300:Copper / Q000032:analysis* / Q000378:metabolism / Q000494:pharmacology; D003304:Coproporphyrinogen Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008670:Metals / Q000032:analysis; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D002614:Chelating Agents; D017931:DNA Primers; D008670:Metals; D011994:Recombinant Proteins; D003300:Copper; D003304:Coproporphyrinogen Oxidase