Structural determinants of cytochrome P450 2B1 specificity: evidence for five substrate recognition sites

Abstract

Twelve site-directed mutants of rat cytochrome P450 2B1 distributed over seven positions and four putative substrate recognition sites (SRS) were constructed and expressed in COS cells. Function was examined using androstenedione and testosterone as substrates. Substitutions at positions 303, 360, and 473 did not markedly affect the regio- or stereoselectivity of androgen metabolism, whereas mutants in positions 206 (SRS-2), 302 (SRS-4), and 363 and 367 (SRS-5) exhibited markedly different steroid metabolite profiles compared with parental P450 2B1. In particular, the Phe-206-->Leu substitution conferred androgen 6 alpha- and testosterone 7 alpha-hydroxylase activities, and the Thr-302-->Ser substitution suppressed androgen 16 beta-hydroxylation in favor of androstenedione 16 alpha- and testosterone 15 alpha-hydroxylation. Replacement of Val-363 or Val-367 with Ala conferred androgen 15 alpha-hydroxylase and 6 beta-hydroxylase activities, respectively, and suppressed susceptibility to mechanism-based inactivation by the P450 2B1-selective chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. The Val-367-->Ala mutant was also resistant to chloramphenicol itself. The Leu mutant at position 363 exhibited increased specificity for androstenedione and testosterone 16 beta-hydroxylation, whereas the Leu mutant at position 367 exhibited decreased stereospecificity. Most interestingly, the size of key residues identified plays a critical role in governing steroid hydroxylation from the alpha-face or beta-face and hydroxylation on the D-ring or the B-ring.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic and Molecular Analysis of 5-Epiaristolochene 1,3-Dihydroxylase, a Cytochrome P450 Enzyme Catalyzing Successive Hydroxylations of Sesquiterpenes

“…In fact, previous studies had already documented the functional significance of this variability. Mutagenesis of the position equivalent to amino acid 368 in several members of the CYP2B subfamily was shown to affect the regiospecific hydroxylation of steroids (52,53). The same position was also reported to play a role in substrate recognition for CYP2A5 (54) and CYP3A4 (55).…”

“…SRS-5 and SRS-6 were of particular interest given their proximity to the active site in several P450 structures (51) and because specific amino acid positions within these regions have previously been correlated with regio-and stereospecific reaction mechanisms (Fig. 4A) (52)(53)(54)(55)(56)(57)(58)(59)(60)(61). A homology model of EAH derived from comparison with the mammalian CYP2C5 structure was constructed (Fig.…”

Threonine-205 in the F Helix of P450 2B1 Contributes to Androgen 16β-Hydroxylation Activity and Mechanism-Based Inactivation

“…Halpert and coworkers have demonstrated that two other F helix residues, Phe-206 and Leu-209, determine the substrate specificity as well as the regioand stereoselectivity of P450 2B1 (He et al, 1994;Szklarz et al, 1995). Similar studies with Phe-209 in P450 2A5 have suggested that this F helix residue plays a critical role in determining substrate and product specificity and that the region around residue 209 constitutes the heme-substrate pocket in mammalian P450s (Lindberg and Negishi, 1989;Juvonen et al, 1991).…”

“…Their studies have identified several residues required for 16␤-hydroxylation of testosterone and androstenedione. For example, mutations of Phe-115 to Ala, Phe-206 to Leu, Leu-209 to Ala, Ser-294 to Ala, Ala-298 to Val, Thr-302 to Ser, and Val-363 to Ala all diminished the 16␤-hydroxylation activity for androgens (He et al, 1994;Szklarz et al, 1995;). Together, , as well as the Thr-205 that has been identified in this study, contribute to the unique characteristics of P450 2B1.…”

“…Residues 114, 302, 363, 367, and 478 have been identified as being in the active site by using mechanism-based inactivators such as secobarbital, N-benzyl-1-aminobenzotriazole, chloramphenicol and N-(2-p-nitrophenethyl)-chlorofluoroacetamide (Kedzie et al, 1991;He et al, 1994He et al, , 1996Kent et al, 1997). The importance of Thr-205 was further emphasized by investigating the susceptibility of the mutant P450s to inactivation by two well characterized mechanism-based inactivators of P450 2B1.…”