Expression of rat heme oxygenase in Escherichia coli as a catalytically active, full-length form that binds to bacterial membranes.

A plasmid, pKK-RHO, was constructed by incorporating the coding sequence of a cDNA for rat heme oxygenase into the expression vector pKK233-2. Escherichia coli strain XL1-blue transformed with pKK-RHO produced a catalytically active, full-length heme oxygenase. The 32-kDa native enzyme expressed, was localized in the bacterial membranes, possibly due to the spontaneous membrane-binding properties of a hydrophobic segment in its C-terminal region. During cultivation, a few degraded forms of heme oxygenase that had lost their membrane-associative properties appeared. Probably, some bacterial proteases cut the native heme oxygenase at sites near its C-terminus and so release hydrophilic peptides of heme oxygenase from the membranes. A 30-kDa polypeptide, one of the degraded forms of heme oxygenase, retained ability to accept electrons from NADPH--cytochrome P450 reductase and also activity for catalyzing breakdown of heme to biliverdin. The cultured cells were pale green. From them we extracted green pigment(s), of which the absorption spectrum closely resembled that of biliverdin, suggesting that a large amount of the endogenous heme of E. coli was actually degraded to biliverdin by the expressed heme oxygenase.