Front Matter

kanes toa,x-dicarboxylic acids (Scheller et al., 1998). Moreover, they observed that

a,x-dicarboxylic acids act as competitive inhibitors ofn-alkane binding (Scheller et

al., 1998).

The mutual exchange of Val in P450 Cm2 and Leu in P450 Alk3A at position 527

led to a direct transposition of the fatty acid chain-length specificities (Zimmer et al.,

1998b), suggesting that amino acids at this site may determine the efficiency of fatty-

acid hydroxylation relatively independent of other active-site residues. Moreover,

Met to Leu substitutions at the corresponding alignment position in P450 Cm1

(CYP52A3), P450 Alk2A (CYP52A5) and P450 Alk5A (CYP52A9) altered the

fatty-acid specificity of these enzymes (Zimmer et al., 1998b). In comparison to

the structure of the bacterial P450 BM-3 (CYP102), it was reported by Zimmer

et al. (1998b) that the amino acid at position 527 may be to close to the sub-

strate-binding pocket near to the heme of CYP52A enzymes, which hydroxylate

fatty acids at thex-position.

18.4.3 TheCYP4 family

The literature reporting fatty acid hydroxylations performed by P450 enzymes of the

familyCYP4 is extensive, and would go beyond the scope of this article. In order to

present this family adequately, general characteristics of theCYP4 family and of a

mainly investigated member, P450 4A1, are discussed in the following paragraphs.

CYP4 is one of the oldest P450 families and contains 22 subfamilies (Simpson,

1997). CYP4 enzymes are primarily involved in hydroxylation of fatty acids, pros-

taglandins, leukotrienes and other eicosanoids in mammalian species (Rendic and Di

Carlo, 1997). The major fatty acid hydroxylating members of theCYP4A subfamily

are summarized in Table 1. These enzymes all show strong preference for hydro-

xylation of the x-position of fatty acids. Another common feature of many

CYP4 enzymes is their inducibility by hypolipidemic agents (Simpson, 1997).

A comparison of the CYP4 enzymes is difficult due to the diversity of experimen-

tal systems employed for activity measurements. The CYP4A enzymes have been

expressed in different hosts such as liver (Baron et al., 1981), kidney (Imaoka et al.,

1993), HepG2 cells (Chaurasia et al., 1995), yeast (Hardwick et al., 1987) or with and

withoutN-terminal modifications inE. coli(Hoch et al., 2000). Additionally, the

conditions for reconstitution of the P450 domain with a reductase part often dif-

fer. A further handicap of these membrane-associated P450 enzymes is that no crys-

tal structure has yet been published, which makes the identification of structure

function relationships difficult. Homology modeling based on the crystal structures

of P450 BM-3 identified some key residues of the CYP4A proteins (Lewis and Lake,

1999). For example, Lewis and Lake (1999) postulated that the ion-pairing to the

fatty acid at position Arg 47 of CYP102 finds its counterpart in CYP4A1 at Lys 93 ,

in CYP4A4 at Lys 90 and in CYP4A11 at Lys 94. For CYP4A1, they further suggested

that the orientation for thex-hydroxylation is achieved via a combination of elec-

trostatic and hydrophobic interactions between substrate and enzyme, involving re-

sidues Lys 93 and Arg 230 necessary for ion-pairing with the carboxyl group of the fatty

acid substrate and the residues Val 118 , Leu 131 , Leu 223 ,Val 385 and Leu 493 , which sup-

18.4 Fatty Acid-hydroxylating P450s Monooxygenases 405