Role of unusual amino acid residues in the proximal and distal heme regions of a plant P450, CYP73A1

CYP73A1 is a typical plant P450 in terms of its function and primary sequen ce. The enzyme catalyzes the rt-hydroxylation of trans-cinnamic acid, the f irst oxidative step in the phenylpropanoid pathway. Its primary protein seq uence exhibits some particular landmarks which are characteristic of plant P450 enzymes. The most interesting is a proline residue (Pro448), very unus ual in animal P450s, just C-terminal to the invariant heme-binding cysteine . To determine the role of this proline, we substituted it with valine, iso leucine, or phenylalanine, residues found in animal P450s, using site-direc ted mutagenesis. Expression of the wild type and mutants in yeast indicated that replacement of Pro448 led to disruption of the heme-protein interacti on, loss of catalytic activity, and either impaired expression or destabili zation of the apoprotein Pro448 is thus essential for the correct insertion of heme in the apoprotein. Another typical feature of CYP73A proteins is t he presence of an alanine-alanine motif (Ala306-Ala307) on the presumed N-t erminal edge of the cleft in the central part of the I helix. This cleft fa ces the iron on the distal. side of the heme and is proposed to be essentia l for catalysis. Substitution of each or both Ala306 and Ala307 residues wi th glycines showed that they are critical for the stability of the protein and influence the positioning of the substrate in the active site. Results are discussed with reference to the structural X-ray data that are availabl e for bacterial P450 proteins.