port or allow hydrophobic interactions to the fatty acid substrate. The CYP4A1 en-
zyme, which was first isolated from rat liver (Tamburini et al., 1984), is one of the
most investigated and active fatty acid-hydroxylating P450 enzymes of theCYP4A
family. Inspired by the natural fusion protein P450 BM-3, Fisher and co-workers
fused the rat liver NADPH reductase to the rat liver P450 4A1. Subsequent expres-
sion of the fusion protein inE. coliresulted in a fatty acid hydroxylation activity
between 10–30 equiv min–1(Fisher et al., 1992a,b). The activity of this fusion
protein was stimulated 10-fold (Chaurasia et al., 1995) and its active site was
mapped by using fatty acid analogs with bulky groups at thex-position and in
the middle of the fatty acid chain (Bambal and Hanzlik, 1996a,b). They showed
that the P450 4A1 fusion protein can hydroxylate lauric acid analogs bearing bulky
groups (e.g.,t-butyl orp-/m-tolyloxy) at thex-position. In contrast, 1,3- or 1,4-phe-
nylene units inserted near the middle of the chain, bind less well and were not
hydroxylated (Bambal and Hanzlik, 1996a, b). For this artificial fusion protein,
NADPH was successfully replaced by electrochemical reduction via the mediator
cobalt(III)sepulchrate resulting in the hydroxylation of lauric acid (Faulkner et
al., 1995).
By the use of a reconstituted system with the human P450 reductase, Hoch and co-
workers (2000) reported high turnover numbers for rat P450 4A1 with values up to
649 equiv min–1.
As shown in Table 5, a strong preference was also found for lauric acid compared
to longer-chain fatty acids. Interestingly, the activity of this rat P450 4A1 depends
strongly on the substrate concentration. An increase in the substrate concentration
from 100lM to 300lM reduces the activity more than 60-fold of this heterologous
inE. coli-expressed P450 4A1 (Hoch et al., 2000).
For P450s of theCYP4A family, the preferred substrates are C 12 –C 20 fatty acids.
Shorter (C 7 –C 10 ) ones are not hydroxylated, or only at a low turnover. These C 7 –C 10
fatty acids are converted by CYP4B1 isoenzymes with turnover numbers between
0.8 and 11 equiv min–1(Fisher et al., 1998). The rabbit P450 4B1 converts not only
fatty acids, but also hydroxylates – as do some members of theCYP52 family –
alkanes such as heptane, octane, nonane and decane at comparable or higher turn-
over numbers (Fisher et al., 1998).
406 18 Fatty Acid Hydroxylations using P450 Monooxygenases
Table 5.Substrate profile and activity of P450 enzymes of the CYP4A1 enzyme.
Fatty acid Turnover[equiv min–1]
(Fatty acid[lM])
Binding constantKsLauric acid 649 (100) 30
Myristic acid 230 (100) 46
Palmitic acid 60 (100) n.m.
Arachidonic acid 6 (80) n.d.
Oleic acid 1.4 (80) n.d.
n.m., not measured; n.d., not detectable