Heterotropic and homotropic cooperativity by a drug-metabolising mutant of cytochrome P450 BM3

• Published in: Biochemical and biophysical research communications Vol. 346; no. 3; pp. 810 - 818
• Main Authors: van Vugt-Lussenburg, Barbara M.A., Damsten, Micaela C., Maasdijk, Dianne M., Vermeulen, Nico P.E., Commandeur, Jan N.M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.08.2006
• Subjects: Acetaminophen - chemistry; Acetaminophen - metabolism; Activation; Allosteric kinetics; Amodiaquine - chemistry; Amodiaquine - metabolism; Arginine - genetics; Arginine - metabolism; Benzoflavones - pharmacology; Biocatalysis; Caffeine - pharmacology; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450 BM3; Dextromethorphan - chemistry; Dextromethorphan - metabolism; Drug metabolism; Enzyme Activation - drug effects; Heterotropic cooperativity; Homotropic cooperativity; Humans; Kinetics; Leucine - genetics; Leucine - metabolism; Molecular Structure; Mutation - genetics; N-Methyl-3,4-methylenedioxyamphetamine - chemistry; N-Methyl-3,4-methylenedioxyamphetamine - metabolism; Pharmaceutical Preparations - metabolism; Phenylalanine - genetics; Phenylalanine - metabolism; Substrate Specificity; Biocatalysis; Homotropic cooperativity; Drug metabolism; Cytochrome P450 BM3; Allosteric kinetics; Activation; Heterotropic cooperativity
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2006.05.179

Recently, we described a triple mutant of the bacterial cytochrome P450 BM3 as the first mutant with affinity for drug-like compounds. In this paper, we show that this mutant, but not wild-type BM3, is able to metabolise testosterone and several drug-like molecules such as amodiaquine, dextromethorphan, acetaminophen, and 3,4-methylenedioxymethylamphetamine that are known substrates of human P450s. Interestingly, the metabolism of 3,4-methylenedioxymethylamphetamine and acetaminophen could be stimulated up to 70-fold by the addition of caffeine, a known activator of rat P450 3A2. With testosterone metabolism, homotropic cooperativity was observed. This shows that heterotropic and homotropic cooperativity, known to occur in the P450 3A family, can also take place in BM3. BM3 therefore can be used as a model system to study atypical kinetics in mammalian P450s. Second, this study shows that BM3 can be engineered to a drug-metabolising enzyme, making it a promising candidate to use as biocatalyst in drug discovery and synthesis.