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 an d in its haem domain. The mutant has been characterized by sequencing, mass spectrometry, steady-state kinetics and by optical and NMR measu rements of substrate binding. The mutant retains significant catalytic activity towards C-12-C-16 fatty acids, catalysing hydroxylation in t he same (omega-1, omega-2, omega-3) positions with k(cat)/K-m values a factor of 14-21 lower. C-12-C-16 alkyl trimethylammonium compounds ar e relatively poor substrates for the wild-type enzyme, but are efficie ntly hydroxylated by the arginine-47 --> glutamate mutant at the omega -1, omega-2 and omega-3 positions, with k(cat) values of up to 19 s(-1 ). Optical spectroscopy shows that the binding of the C-14 and C-16 al kyl trimethylammonium compounds to the mutant is similar to that of th e corresponding fatty acids to the wild-type enzyme. Paramagnetic rela xation measurements show that laurate binds to the ferric state of the mutant in a significantly different position, 1.5 Angstrom closer to the iron, than seen in the wild-type, although this difference is much smaller (similar to 0.2 Angstrom) in the ferrous state of the complex . The binding of a substrate having the same charge as residue 47 to t he 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 i on-pair interaction with this residue). The results are discussed in t he light of the three-dimensional structure of the enzyme.