Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds.
Oligonucleotide-directed mutagenesis has been used to replace arginine-47 with glutamate in cytochrome P-450 BM3 from Bacillus megaterium and in its haem domain. The mutant has been characterized by sequencing, mass spectrometry, steady-state kinetics and by optical and NMR measurements of substrate binding. The mutant retains significant catalytic activity towards C12-C16 fatty acids, catalysing hydroxylation in the same (omega-1, omega-2, omega-3) positions with kcat/Km values a factor of 14-21 lower. C12-C16 alkyl trimethylammonium compounds are relatively poor substrates for the wild-type enzyme, but are efficiently hydroxylated by the arginine-47-->glutamate mutant at the omega-1, omega-2 and omega-3 positions, with kcat values of up to 19 s-1. Optical spectroscopy shows that the binding of the C14 and C16 alkyl trimethylammonium compounds to the mutant is similar to that of the corresponding fatty acids to the wild-type enzyme. Paramagnetic relaxation measurements show that laurate binds to the ferric state of the mutant in a significantly different position, 1.5 A closer to the iron, than seen in the wild-type, although this difference is much smaller ( approximately 0.2 A) in the ferrous state of the complex. The binding of a substrate having the same charge as residue 47 to the ferric state of the enzyme is roughly ten times weaker than that of a substrate having the opposite charge (and thus is able to make an ion-pair interaction with this residue). The results are discussed in the light of the three-dimensional structure of the enzyme.
Oliver
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Centre for Mechanisms of Human Toxicity, P.O. Box 138, Hodgkin Building, Lancaster Road, Leicester LE1 9HN, U.K. 7RH, U.K. and Biological NMR Centre, Medic.
Modi
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Primrose
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Lian
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Roberts
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D019943:Amino Acid Substitution; D001120:Arginine; D001410:Bacillus megaterium / Q000201:enzymology*; D001426:Bacterial Proteins; D001665:Binding Sites; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D018698:Glutamic Acid; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase; D019906:Nuclear Magnetic Resonance, Biomolecular; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005230:Fatty Acids, Nonesterified; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D018698:Glutamic Acid; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases