Heterotropic and homotropic cooperativity by a drug-metabolising mutant of cytochrome P450 BM3.
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.
van Vugt-Lussenburg
Barbara M A
BM
LACDR/Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Damsten
Micaela C
MC
Maasdijk
Dianne M
DM
Vermeulen
Nico P E
NP
Commandeur
Jan N M
JN
D000082:Acetaminophen / Q000737:chemistry / Q000378:metabolism; D000655:Amodiaquine / Q000737:chemistry / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001571:Benzoflavones / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003915:Dextromethorphan / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics*; D018817:N-Methyl-3,4-methylenedioxyamphetamine / Q000737:chemistry / Q000378:metabolism; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D001571:Benzoflavones; D004364:Pharmaceutical Preparations; D000655:Amodiaquine; D000082:Acetaminophen; D002110:Caffeine; D010649:Phenylalanine; C011512:alpha-naphthoflavone; D003915:Dextromethorphan; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D007930:Leucine; D018817:N-Methyl-3,4-methylenedioxyamphetamine