Cytochrome P450 active site plasticity: attenuation of imidazole binding in cytochrome P450cam by an L244A mutation.

the imidazole coordinates tothe iron in an orientation roughly perpendicular to theplane of the heme, in the L244A mutant the rearranged Ihelix, and specifically residue Val247, forces the imidazoleinto an orientation almost parallel to the heme that impairsits ability to coordinate to the heme iron. As a result, theimidazoleismuchmoreweaklyboundtothemutantthanitis to the wild-type enzyme. Despite the constriction of theactive site by the mutation, previous work with the L244Amutanthasshownthatitoxidizeslargersubstratesthanthewild-type enzyme. This paradoxical situation, in which amutation that nominally increases the active site cavityappears to decrease it, suggests that the mutation actuallyincreases the active site maleability, allowing it to betterexpand to oxidize larger substrates.Keywords: active site conformation/azole drug resistance/cytochrome P450 inhibition/imidazole binding to P450/protein maleabilityIntroductionAzole-based compounds such as ﬂuconazole, ketoconazoleand itraconazole are potent inhibitors of cytochrome P450enzymes (Little and Ryan, 1982; Loose et al., 1983; Murrayand Ryan, 1983; Benedetti and Bani, 1999) and are employedin the treatment of topical and systemic fungal infections(Keating, 2005; Krcmery, 2005; Lacroix et al., 2005). Thepotentialutilityofazolesinotherclinicalcontexts,forexamplein therapeutic approaches to tuberculosis (Guardiola-Diazet al., 2001; McLean et al., 2002), has also received atten-tion. However, azole-resistant fungal strains are now wellknown (Asai et al., 1999; Kelly et al., 1999; Cuenca-Estrella et al., 2005; Meneau and Sanglard, 2005). An under-standing of the parameters that control the binding of azolesto hemoproteins is thus relevant to the development of azoleagents that circumvent resistance, possess greater speciﬁcityfor particular hemoproteins or address new targets. Becauseazole compounds are still the primary antifungal therapeuticsincurrentuse,anunderstandingofazolebindingandresistanceis particularly critical in this area.Until recently, structural insights into the origins of mit-igated azole afﬁnity were derived primarily from homologymodels and experiments with model P450 enzymes, becauseno crystal structure existed of a therapeutically relevant target.The crystallization of CYP51 from Mycobacterium tubercu-losis in 2001 provided the ﬁrst structurally characterizedpotential drug target in the P450 family, as well as a crys-tallographic model for the CYP51 enzymes that are the actualtargets in fungi (Podust et al., 2001). Azole-based compoundshave been shown to bind and inhibit CYP51 (Podust et al.,2001; Matsuura et al., 2005). The insights derived from homo-logy models of resistant strains of CYP51 revealed that muta-tions near the heme cofactor are likely to be responsible for thealtered afﬁnity of azole-based agents (Sheng et al., 2004).In previous work, we constructed the L244A mutant ofP450cam based on the hypothesis that the active site would bemodestly expanded relative to that of the wild-type enzyme,because the alanine side chain is three carbons smaller thanthat of the normal leucine (De Voss et al., 1997). In accordwith this hypothesis, and with the predictions of an associatedinsilicodockingstudy,theL244Amutantwasfoundtooxidizelarger molecules than the wild-type enzyme. More recently,we reported a computational docking effort to predict thedifferential binding of substituted imidazoles to the activesites of cytochrome P450