The Amino Acid Residues Affecting the Activity and Azole Susceptibility of Rat CYP51 (Sterol 14-Demethylase P450)

Abstract

The amino acid residues affecting the function of rat sterol 14-demethylase P450 (CYP51) were examined by means of point mutation. Forty-five mutants with respect to 27 amino acid sites were constructed and expressed in Escherichia coli. Substitution of highly conserved Y131, E369, R372, or R382 decreased the expression of CYP51 protein, indicating some structural importance of these residues. Substitution of H314, T315, or S316 caused considerable effects on the catalytic activity, and T315 was identified as the "conserved threonine" of CYP51. H314 was important for maintenance of the activity of CYP51 and was a characteristic residue of this P450, because the position corresponding to this residue is occupied by an acidic amino acid in most other P450 species. A144 was identified as a residue affecting the interaction of CYP51 with ketoconazole. Substitution of A144 with I, which occupies the corresponding position in fungal CYP51, enhanced the ketoconazole susceptibility of rat CYP51 with little change in the catalytic activity, indicating an important role of this residue in determination of the ketoconazole susceptibility of CYP51. Alteration of the catalytic activity was caused by the substitution at some other sites, whereas substitution of a few highly conserved amino acids caused little alteration of the activity of CYP51.

Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Δ7-24, 25-dihydrolanosterol

“…The enzyme-sterol complex supports this notion, clearly showing that SRS4 in the CYP51 structure is actually located further upstream in the I-helix (residues M284-290 in Table 2 ). Interestingly, mutation of the corresponding T in rat CYP51 to A, although signifi cantly (6-fold) decreasing enzymatic activity, does not abolish it completely, whereas its substitution to bulkier residues (R/N) makes the activity undetectable ( 57 ). This suggests that, provided there is enough space for the catalytic water molecule in this area, the CYP51 I-helix hydrogen bond network can partially compensate for the lack of the threonine hydroxyl in the mechanism of the proton delivery.…”

Sterol 14α-demethylase cytochrome P450 (CYP51), a P450 in all biological kingdoms

“…SRS1 (B' helix/B'/C loop) forms the upper surface of a P450 substrate binding cavity ( Figure 5). Substitution of the conserved Y, F and G in the B' helix results in complete loss of sterol 14α-demethylase activity of M. tuberculosis and human CYP51 [42] and was shown to strongly affect activity of the rat ortholog (the corresponding Y131 and F139 were studied [46]). Another example provides mutation of conserved P81 in M. tuberculosis CYP51 (there is always P in this position with the exception of one sequence from filamentous fungi Cunninghamella elegans (Y)).…”

“…Mammalian steroidogenic CYPs (e.g. CYPs 7,8,11,17,19,21,27,39,46) might be another example of this type of diversification. Sterol metabolizing P450s often preserve narrow substrate specificity and among them there are other examples which catalyze multiple step reactions, e.g.…”

“…This region contains the unique triplet of the CYP51 family -HT/sS-. The role of each of these three residues has been thoroughly studied on rat CYP51 [46,49]. The first and the last residues in the triplet (Figure 4) are conserved throughout the CYP51 family, while the middle T, corresponding to the "conserved threonine" [50] in most P450s, is replaced with S in several sequences from filamentous fungi.…”

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

“…We searched the databases for SNPs in the human population causing missense mutations in any of the known regions important for substrate recognition, enzymatic activity, POR interaction and azole binding (Nitahara et al, 2001;Lepesheva et al, 2003;Strushkevich et al, 2010). We also compared existing SNPs to previously published in vitro mutations of human and rat CYP51A1 and proposed a potential effect of such mutation in humans ( Table 2).…”