The dimerization of Pseudomonas putida cytochrome P450cam:: practical consequences and engineering of a monomeric enzyme

Cytochrome P450(cam) dimerizes via the formation of an intermolecular disulfide bond, complicating the storage and handling of the enzyme, particularly at higher concentrations. The dimeric enzyme is 14% less active than the monomer and forms at a slow but significant rate even at 4 degrees C (k = 1.09 x 10(-3) mM(-1) h(-1)). To eliminate any ambiguity introduced by dimer formation and to simplify handling and storage of the enzyme, site-directed mutagenesis was used to identify C334 as the single cysteine residue responsible for the formation of the disulfide linkage and to engineer a monomeric enzyme by substituting an alanine in its place. The C334A mutant is identical with the wild-type P450(cam) monomer in terms of optical spectra, camphor binding and turnover activity, but shows no evidence of dimerization and aggregation even at millimolar concentrations. Preliminary H-1 NMR investigations also indicate a significant improvement in the quality of spectra obtained with this mutant. (C334A)P450(cam) is therefore proposed as an alternative to the wild-type enzyme-a base mutant otherwise identical with the wild-type but with improved handling characteristics.