The primary sequence of cytochrome P450tyr, the multifunctional N-hydroxylase catalyzing the conversion of L-tyrosine to p-hydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.

The heme thiolate protein cytochrome P450tyr is a multifunctional N-hydroxylase converting L-tyrosine to p-hydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (Sibbesen et al. (1995) J. Biol. Chem. 270, 3506-3511). Using a polyclonal antibody toward cytochrome P450tyr and oligonucleotide probes designed on the basis of amino acid sequences of tryptic fragments, a full-length cDNA clone encoding cytochrome P450tyr has been isolated and sequenced. The open reading frame encodes a protein with a molecular mass of 61,887 Da. A comparison with the amino acid sequencing data demonstrates that the protein is not subjected to posttranslational modification at the N- and C-terminal ends except for the removal of the N-terminal methionine residue. Highest positional identity (30.8%) is found to the 3',5'-flavonoid hydroxylase of petunia (CYP75A1) and to a cytochrome P450 sequence from avocado of unknown function (CYP71A1). Consequently, cytochrome P450tyr is assigned as the first member of a new cytochrome P450 family denoted CYP79. The N-terminal region of cytochrome P450tyr contains the four domains characteristic for cytochrome P450 enzymes of the endoplasmic reticulum (ER) in animals. The amino acid sequence before the proline-rich domain is longer in cytochrome P450tyr and in four cytochrome P450s presently available from other monocotyledoneous plants compared to the sequences from dicotyledoneous plants but is concluded to contain a single transmembrane helix with the N-terminal located in the lumen of the ER and the bulk of the protein protruding into the cytoplasm. The heme-binding cysteine residue of cytochrome P450tyr is recognizable at position 493 but this region deviates from the consensus sequence by having an unusual alanine residue at position 495. The central region of helix I contains three residues, Ala-352, Asn-355, and Pro-356, deviating from the consensus sequence. CYP56 is the only other known cytochrome P450 using tyrosine as substrate and contains the same Asn-Pro substitution in the consensus sequence of helix I indicating the importance of these residues in defining substrate specificity. The conserved threonine residue which normally helps to form the oxygen binding pocket is absent. The cytochrome P450tyr sequence represents the first amino acid sequence of a functionally characterized cytochrome P450 enzyme from a monocotyledoneous plant and the first sequence of a membrane-bound N-hydroxylase with high substrate specificity. Multifunctional N-hydroxylases of the cytochrome P450 type have not been previously demonstrated to catalyze biosynthetic pathways in living organisms.