Xenopus laevis CYP17 Regulates Androgen Biosynthesis Independent of the Cofactor Cytochrome b5

Abstract

The enzyme CYP17 primarily regulates androgen production by mediating four reactions: conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively (17␣-hydroxylase activity), followed by conversion of the 17-hydroxylated steroids to dehydroepiandrosterone and androstenedione, respectively (17,20-lyase activity). Most mammalian CYP17 isoforms have high 17␣-hydroxylase relative to 17,20-lyase activities and preferentially mediate one of the two 17,20-lyase reactions. In contrast, Xenopus laevis CYP17 potently regulates all four reactions in the frog ovary. CYP17 isoforms generally rely on the cofactor cytochrome b 5 for the 17,20-lyase reaction, suggesting that the high lyase activity of Xenopus CYP17 might be due to a lesser dependence on b 5 . The kinetics of Xenopus CYP17 expressed in yeast microsomes were therefore examined in the absence and presence of Xenopus on human b 5 . Xenopus CYP17 mediated both 17,20-lyase reactions in the absence of b 5 , confirming that the activity did not require b 5 . However, both Xenopus and human b 5 slightly enhanced Xenopus CYP17-mediated lyase activity, indicating that the enzyme was still at least partially responsive to b 5 . Surprisingly, only the human b 5 cofactor enhanced human CYP17-mediated lyase activity, implying that the human enzyme had more specific cofactor requirements than Xenopus CYP17. Studies using human/Xenopus chimeric b 5 proteins revealed that human b 5 residues 16 -41 were important for the specific regulation of the lyase activity of HuCYP17, possibly serving as an interacting domain with the enzyme. CYP17 may therefore have evolved from a general producer of sex steroids in lower vertebrates to a more tightly regulated producer of both sex steroids and glucocorticoids in mammals.The development and maintenance of male and female fertility depends on the normal function of androgens and their cognate androgen receptor. Androgens induce male sexual differentiation before birth, promote sexual maturation during puberty, and maintain male reproductive function in adults (1). In women, androgens serve as precursors for estrogen biosynthesis (2) while also playing significant roles in normal as well as pathologic ovarian development (3-8).Androgen production depends on the enzyme CYP17 (9). CYP17 mediates two enzymatic reactions. Its 17-hydroxylase activity primarily converts pregnenolone (⌬5 pathway) and progesterone (⌬4 pathway) to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively. The 17,20-lyase activity of CYP17 then converts the 17-hydroxylated pregnenolone and progesterone to dehydroepiandrosterone (DHEA) 1 and androstenedione, respectively. Notably, the cofactor cytochrome b 5 has been implicated as a critical component of the 17,20-lyase reaction. Nearly all mammalian CYP17 isoforms that have been examined have little 17,20-lyase activity in the absence of b 5 . The addition of b 5 markedly enhances lyase activity, although it usually remains lower than the hydroxylase activity und...

Cytochrome b5 modulation of 17α hydroxylase and 17–20 lyase (CYP17) activities in steroidogenesis

“…Interestingly, a CYP17 from Xenopus laevis displayed unusually high lyase activity in the absence of either frogor human-derived cytochrome b 5 (Yang & Hammes 2005). Moreover, the rate of cleavage activity of Xenopus laevis CYP17 in the absence of cytochrome b 5 was found to be similar to human CYP17 assayed in the presence of human cytochrome b 5 .…”

“…The identity of such residues on the surface of cytochrome b 5 may similarly be vital for CYP17 cleavage. More recently, residues 16-41 in human cytochrome b 5 were found to be necessary in the specific regulation of the lyase activity of human CYP17, possibly serving as an interacting domain with the enzyme (Yang & Hammes 2005). Indeed, further detailed identification of the key residues for CYP17 and cytochrome b 5 interaction will give insight into the nature of interaction between cytochrome b 5 and CYP17 and shed light on how cytochrome b 5 is able to promote a subtle conformational change of CYP17.…”

Comparison of the 17α-Hydroxylase/C17,20-Lyase Activities of Porcine, Guinea Pig and Bovine P450c17 Using Purified Recombinant Fusion Proteins Containing P450c17 Linked to NADPH-P450 Reductase

“…It is interesting to note that Xenopus P450c17 exhibits similar rates of hydroxylation of P4 and cleavage of 17αOH P4 as the mammalian enzymes presented here while metabolism of P5 is much lower than P4 by comparison and more typical of the guinea pig enzyme. However, unlike C17,20-lyase activities of the porcine, guinea pig, and bovine enzymes that were similarly stimulated, 3 to 5-fold, in the presence of b 5 , the Xenopus C17,20-lyase activity exceeded the rate of 17α-hydroxylation and was not stimulated by b 5 (Yang and Hammes, 2005). Therefore, it will be of interest to extend the current study to other P450c17s to determine if specific patterns of amino acid sequences can be distinguished relating differences in enzymatic properties with differences in their amino acid sequences.…”

Effect of perfluorooctanesulfonate exposure on steroid hormone levels and steroidogenic enzyme activities in juvenile Silurana tropicalis

“…The OH-P and AD concentrations in the assay medium were measured initially and following the reaction with ELISA as described above. CYP17 17,20-lyase activity was confirmed in accordance with the methods of Auchus, Lee and Miller (1998) and Yang and Hammes (2005) with modifications. For the measurement of CYP17 17hydroxylase and 17,20-lyase activity, approximately 0.5 g of gonad tissue was homogenized in cold 0.05 M Tris buffer (pH 7.4).…”

“…(2) a 17,20-lyase activity, which metabolizes the 17-hydroxylated progestins to dehydroepiandrosterone and androstenedione (AD), respectively. Yang and Hammes (2005) have shown that sexually mature X.…”