activity of P450 BM-3 (Boddupalli et al., 1992). Ifx-oxo fatty acids, such as 18-
oxooctadecanoic, 16-oxohexadecanoic, 14-oxotetradecanoic and 12-oxododecanoic
acids, are used as substrates, the correspondinga,x-diacids are generated (Davis et
al., 1996).
Oliver and co-worker (1997a,b) showed that the amino acid at position 87 mod-
ulates the regioselectivity of the fatty acid hydroxylation. A single mutation
(Phe87Ala) shifted the regioselectivity for lauric and myristic acid from subterminal
to nearly exclusive terminal hydroxylation. An exchange of Phe 87 to Val converted
cytochrome P450 BM-3 into a regio- and stereoselective (14S,15R)-arachidonic acid
epoxygenase (Graham-Lorence, 1997).
Replacement of Arg 47 with Glu resulted in the ability of this P450 BM-3 mutant to
hydroxylateN-alkyltrimethylammonium compounds (Table 2), which was explained
by an inversion of the substrate binding conditions (Oliver et al., 1997a). P450 BM-3,
heterologously expressed inE. coli, has been usedin vivoto produce mixtures of
chiral 12-, 13- and 14-hydroxypentadecanoic acid in preparative scale at high op-
tical purity (Schneider et al., 1998). Furthermore, P450 BM-3 and its mutant
Phe 87 Ala can be expressed in gram scale and efficiently purified in a single step
for further enzyme-based biotransformation reactions in preparative scale (Schwa-
neberg et al., 1999b). The pNCA activity assay shown in Figure 4 allows the deter-
mination of the P450 BM-3 wild-type and mutant F87A activity without background
reaction (Schwaneberg et al., 1999a). After the hydroxylation of the terminal posi-
tion, an unstable hemiacetal is produced which spontaneously dissociates into thex-
oxo-carboxylic acid and the chromophorep-nitrophenolate. The latter can be easily
quantified at 410 nm using a spectrophotometer.
The pNCA assay is amenable to automation (Schwaneberg et al., 1999a), and is
usable in a high-throughput screening environment to identify P450 BM-3 variants
with beneficial mutants to overcome the deficiencies of the P450 BM-3 enzyme with
respect to temperature, organic solvent or pH stability.
18.4 Fatty Acid-hydroxylating P450s Monooxygenases 403
Figure 4. Principle of the colorimetric pNCA assay allowing the determination of the fatty acid hydro-
xylating activity of P450 BM-3 fromBacillus megateriummutant F87A (Schwaneberg et al., 1999a).