Cloning and Expression of Cytochrome P450 Enzymes Catalyzing the Conversion of Tyrosine to p-Hydroxyphenylacetaldoxime in the Biosynthesis of Cyanogenic Glucosides in Triglochin maritima

• Published in: Plant physiology (Bethesda) 2000-04, Vol.122 (4), p.1311-1321
• Main Authors: Nielsen, J S, Møller, B L
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Biochemistry and Macromolecular Structure; Biological and medical sciences; Biosynthesis; Catalysis; Cloning, Molecular; Codons; Complementary DNA; CYP79A1 protein; CYP79E1 protein; CYP79E2 protein; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochromes; DNA, Complementary; Enzymes; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Glucosides; Glucosides - biosynthesis; Metabolism; Molecular Sequence Data; Oximes - metabolism; p-hydroxymandelonitrile; p-Hydroxyphenylacetaldoxime; Plant physiology and development; Poaceae - metabolism; Polymerase Chain Reaction; Sequence Homology, Amino Acid; Sorghum; Sorghum bicolor; taxiphyllin; Triglochin maritima; triglochinin; Tyrosine; Tyrosine - metabolism
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.122.4.1311

Two cDNA clones encoding cytochrome P450 enzymes belonging to the CYP79 family have been isolated from Triglochin maritima. The two proteins show 94% sequence identity and have been designated CYP79E1 and CYP79E2. Heterologous expression of the native and the truncated forms of the two clones in Escherichia coli demonstrated that both encode multifunctional N-hydroxylases catalyzing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the two cyanogenic glucosides taxiphyllin and triglochinin in T. maritima. This renders CYP79E functionally identical to CYP79A1 from Sorghum bicolor, and unambiguously demonstrates that cyanogenic glucoside biosynthesis in T. maritima and S. bicolor is catalyzed by analogous enzyme systems with p-hydroxyphenylacetaldoxime as a free intermediate. This is in contrast to earlier reports stipulating p-hydroxyphenylacetonitrile as the only free intermediate in T. maritima. L-3,4-Dihydroxyphenyl[3-14C]Ala (DOPA) was not metabolized by CYP79E1, indicating that hydroxylation of the phenol ring at the meta position, as required for triglochinin formation, takes place at a later stage. In S. bicolor, CYP71E1 catalyzes the subsequent conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile. When CYP79E1 from T. maritima was reconstituted with CYP71E1 and NADPH-cytochrome P450 oxidoreductase from S. bicolor, efficient conversion of tyrosine to p-hydroxymandelonitrile was observed.