Raspberry ketone is an important compound for the flavour industry. It is frequently used in products such as soft drinks, sweets, puddings and ice creams. The compound can be produced by organic synthesis. Demand for "natural" raspberry ketone is growing considerably. However, this product is extremely expensive. Consequently, there is a remaining desire to better understand how raspberry ketone is synthesized in vivo, and which genes and enzymes are involved. With this information we will then be in a better position to design alternative production strategies such as microbial fermentation. This article focuses on the identification and application of genes potentially linked to raspberry ketone synthesis. We have isolated candidate genes from both raspberry and other plants, and these have been introduced into bacterial and yeast expression systems. Conditions have been determined that result in significant levels of raspberry ketone, up to 5 mg/L. These results therefore lay a strong foundation for a potentially renewable source of "natural" flavour compounds making use of plant genes.

Cytochrome P-450TYR, which catalyzes the N-hydroxylation of L-tyrosine in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench has recently been isolated (Sibbesen, O., Koch, B., Halkier, B. A., and Møller, B. L. (1994) Proc. Natl. Acad. Sci. U.S.A. 92, 9740-9744). Reconstitution of the enzyme activity in lipid micelles containing cytochrome P-450TYR and NADPH-cytochrome P-450 oxidoreductase demonstrates that cytochrome P-450TYR catalyzes the conversion of L-tyrosine into p-hydroxyphenylacetaldehyde oxime. Earlier studies with microsomes have demonstrated that this conversion involves two N-hydroxylation reactions of which the first produces N-hydroxytyrosine. We propose that the product of the second N-hydroxylation reaction is N,N-dihydroxytyrosine. N,N-dihydroxytyrosine is dehydrated to 2-nitroso-3-(p-hydroxyphenyl) propionic acid which decarboxylates to p-hydroxyphenylacetaldehyde oxime. The dehydration and decarboxylation reactions may proceed non-enzymatically. The E/Z ratio of the p-hydroxyphenylacetaldehyde oxime produced by reconstituted cytochrome P-450TYR is 69:31. Lipid micelles made from L-alpha-dilauroyl phosphatidylcholine are more than twice as effective in reconstituting cytochrome P-450TYR activity as compared to other lipids. The Km and turnover number of the enzyme is 0.14 mM and 200 min-1, respectively, when assayed in the presence of 15 mM NaCl whereas the values are 0.21 mM and 230 min-1 when assayed in the absence of added salt. The multifunctional nature cytochrome P-450TYR is confirmed by demonstrating that binding of L-tyrosine or N-hydroxytyrosine mutually excludes binding of the other substrate. These results explain why the conversion of tyrosine to p-hydroxyphenylacetaldehyde oxime as earlier reported (Møller, B. L., and Conn, E. E. (1980) J. Biol. Chem. 255, 3049-3056) shows the phenomenon of catalytic facilitation ("channeling"). Cytochrome P-450TYR is the first isolated multifunctional heme-thiolate enzyme from plants. N-Hydroxylases of the cytochrome P-450 type with high substrate specificity have not previously been reported.

The substrate specificity of cytochrome P450, defined as the ability of a compound to promote NAD(P)H and O 2 utilization in the production of either organic or reduced oxygen metabolites, is largely determined by steric and hydrophobic interactions. P450 specificity may therefore be determined by the "fit" of a compound within the active site. A receptor-constrained three-dimensional screening program (DOCK) has been used to select 11 compounds predicted to fit within the P450 cam active site and 5 compounds predicted to fit within the L244A P450 cam but not wild-type active site. The 16 compounds were evaluated as P450 cam substrates by measuring (a) binding to the enzyme, (b) stimulation of NADH and O 2 consumption, (c) enhancement of H 2 O 2 production, and (d) formation of organic metabolites. Seven of the compounds predicted to fit in the active site, and none of the compounds predicted not to fit, were found to be substrates. Compounds predicted to fit very tightly within the active site were poor or non-substrates. The L244A P450 cam mutant was constructed, expressed, purified, and shown to readily oxidize some of the larger compounds incorrectly predicted to be substrates for the wild-type enzyme. The 5 ligands selected to fit the L244A but not wild-type sites were not detectable substrates, presumably because they fit too tightly into the active site. Retroactive adjustments of the docking program based on an analysis of the docking parameters, particularly variation of the minimum distance allowed between ligand and protein atoms, allow correct predictions for the activity of 15 of the 16 compounds with wild-type P450 cam . The DOCK predictions for the L244A mutant were also improved by changing the minimum contact distances to disfavor the larger compounds. The results indicate that ligands that fill the active site are marginal or non-substrates. A degree of freedom of motion is required for substrate positioning and catalytic function. If parameters are chosen to allow for this requirement, P450 cam substrate predictions based on ligand docking in the active site can be reasonably accurate.

Fusion proteins of cytochrome P450cam with putidaredoxin (Pd) and putidaredoxin reductase (PdR), the two proteins required to transfer electrons from NADH to P450cam, were constructed by fusing cDNAs encoding the three proteins in the expression vector pCWori+. Several fusion proteins, in which the order of the three protein domains and the linkers between them were varied, were expressed in Escherichia coli, purified, and characterized. The highest activity (kcat = 30 min-1) was obtained with a PdR-Pd-P450cam construct in which the peptides TDGTASS and PLEL were used, respectively, to link the PdR to the Pd and the Pd to the P450cam domains. Oxygen and NADH consumption is tightly coupled to substrate oxidation in the fusion proteins. The rate-limiting step in the catalytic turnover of these fusion proteins is electron transfer from Pd to P450cam. This is indicated by high rates of electron transfer from the PdR and Pd domains to exogenous electron acceptors, by an increase in the activity of the P450cam domain upon addition of exogenous Pd, and by the high activity of wild-type P450cam when incubated with a PdR-Pd fusion protein. E. coli cells expressing the PdR-Pd-P450cam fusion protein efficiently oxidize camphor to 5-exo-hydroxycamphor and 5-oxocamphor. E. coli cells expressing the triple fusion protein thus constitute the first heterologous self-sufficient catalytic system for the oxidation of camphor and other substrates by P450cam.

The cytochrome P-450 enzyme (hemethiolate enzyme) that catalyzes the N-hydroxylation of L-tyrosine to N-hydroxytyrosine, the committed step in the biosynthesis of the cyanogenic glucoside dhurrin, has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The cytochrome P-450 enzyme was solubilized with the detergents Renex 690, reduced Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and isolated by ion-exchange (DEAE-Sepharose) and dye (Cibacron blue and reactive red 120) column chromatography. To prevent irreversible aggregation of the cytochrome P-450 enzyme, the isolation procedure was designed without any concentration step--i.e., with dilution of the ion-exchange gel with gel filtration material. The isolated enzyme, which we designate the cytochrome P-450TYR enzyme, gives rise to the specific formation of a type I substrate binding spectrum in the presence of L-tyrosine. The microsomal preparation contains 0.2 nmol of total cytochrome P-450/mg of protein. The cytochrome P-450TYR enzyme is estimated to constitute approximately 20% of the total cytochrome P-450 content of the microsomal membranes and about 0.2% of their total protein content. The apparent molecular mass of the cytochrome P-450TYR enzyme is 57 kDa, and the N-terminal amino acid sequence is ATMEVEAAAATVLAAP. A polyclonal antibody raised against the isolated cytochrome P-450TYR enzyme is specific as monitored by Western blot analysis and inhibits the in vitro conversion of L-tyrosine to p-hydroxymandelonitrile catalyzed by the microsomal system. The cytochrome P-450TYR enzyme exhibits high substrate specificity and acts as an N-hydroxylase on a single endogenous substrate. The reported isolation procedure based on dye columns constitutes a gentle isolation method for cytochrome P-450 enzymes and is of general use as indicated by its ability to separate cytochrome P-450TYR from the cytochrome P-450 enzyme catalyzing the C-hydroxylation of p-hydroxyphenylacetonitrile and from cinnamic acid 4-hydroxylase.