Fatty acids, alcohols, amides and other compounds which are able to establish
hydrogen bonds with Arg 47 and/or Thr 51 will be hydroxylated. Esters and nonpolar
compounds such as alkanes, which are unable to create these interactions or which
cannot path the “hydrophobic mouth” at the beginning of the substrate access chan-
nel, are not or only marginally converted by P450 BM-3. Regioselectivity of the
subterminal fatty acid hydroxylase P450 BM-3 depends strongly on the chain-
length of the fatty acid substrate (Table 3).
Truan and co-workers (Truan et al., 1999) determined the absolute configuration
of three primary metabolites obtained from palmitic acid showing high enantiomeric
excess for (R)-15- and (R)-14-hydroxypalmitic acid (both 98 % ee) and somewhat
lower selectivity forx-3 hydroxylation (72 % ee) yielding (R)-13-hydroxypalmitic
acid. Arachidonic acid is metabolized by P450 BM-3 to nearly enantiomerically pure
(R)-18-hydroxyeicosatetraenoic acid (80 % of total products) and 14(S),15(R)-
epoxyeicosatrienoic acid (20 % of total products). The arachidonic acid analogs
eicosapentaenoic acid and eicosatrienoic acids were converted quantitatively to
17(S),18(R)-epoxyeicosatetranoic acid or 17-, 18-, and 19-hydroxyeicosatrienoic
acid in a ratio of 2.4 : 2.2 : 1 (Capdevila et al., 1996). In addition to hydroxyla-
tion, Boddupalli and co-workers observed the formation of ketones such as 14-
oxo- and 13-hydroxy-14-oxo-palmitic acid, which was related to a dehydrogenase
402 18 Fatty Acid Hydroxylations using P450 Monooxygenases
Table 3.Regioselectivity of P450 BM-3 catalyzed hydroxylations of aliphatic substrates (Miura and
Falco, 1975).
Substrate Chain-length Distribution of hydroxyl isomersa[%]
x-1 x-2 x-3Fatty acid 12 36 30 34
13 17 65 18
14 44 28 2815 32 49 19
16 31 48 21
17 49 35 1618 39 47 14Alcohol 12 22 51 27
13 24 49 27
14 30 44 2615 27 54 19
16 22 41 37Amide 12 21 45 34
14 34 34 3215 28 57 15
16 29 45 26amajor products are highlighted in bold numbers