Arlt Wiebke, Martens John W M, Song Maengseok, Wang Jonathan T, Auchus Richard J, Miller Walter L
Department of Pediatrics and the Metabolic Research Unit, University of California, San Francisco, California 94143-0978, USA.
Endocrinology. 2002 Dec;143(12):4665-72. doi: 10.1210/en.2002-220456.
Adrenarche is the prepubertal onset of increased adrenal secretion of 19-carbon steroids, especially dehydroepiandrosterone (DHEA). However, while human beings and chimpanzees exhibit adrenarche, other primates such as the baboon and rhesus monkey do not, and the adrenals of most other mammals produce little or no DHEA. Thus, the acquisition of adrenarche is a very recent evolutionary event. DHEA is produced from pregnenolone by the successive 17alpha-hydroxylase and 17,20 lyase activities of a single enzyme, P450c17. To ascertain whether sequence differences in P450c17 contribute to adrenarche, we cloned the rhesus monkey cDNA from adrenal tissue and cloned the chimpanzee and baboon cDNAs from genomic DNA using an exon-trapping strategy. Using microsomes from yeast transformed with rhesus, baboon, chimp, or human P450c17, we measured the Michaelis constant and maximum velocity for the 17alpha-hydroxylase and 17,20 lyase activities. The human and chimp enzymes differ at only two amino acids and baboon and rhesus P450c17 only at a single residue; the human/chimp enzyme differed from the baboon/rhesus enzyme by 25-27 residues (95% identity). Surprisingly, the greatest difference in enzymatic activities was a marked increase in 17alpha-hydroxylase activity of P450c17 in the baboon, which differs from rhesus only at residue 255 [arginine (Arg) in baboon, histine (His) in rhesus]. Residue 255 is also Arg in human and chimp. Wild-type human P450c17 and its Arg255His mutant had similar 17alpha-hydroxylase activities, but the Arg255Ala mutant had decreased 17alpha-hydroxylase activity. These data establish that Arg255 is important for 17alpha-hydroxylase activity and show that the evolution of adrenarche in higher primates is not determined by variations in the sequence of P450c17.
肾上腺初现是青春期前肾上腺分泌的19碳类固醇增加的起始阶段，尤其是脱氢表雄酮（DHEA）。然而，人类和黑猩猩会出现肾上腺初现，而其他灵长类动物如狒狒和恒河猴则不会，并且大多数其他哺乳动物的肾上腺几乎不产生或不产生DHEA。因此，肾上腺初现的出现是一个非常近期的进化事件。DHEA由孕烯醇酮通过单一酶P450c17的连续17α-羟化酶和17,20裂解酶活性产生。为了确定P450c17中的序列差异是否导致肾上腺初现，我们从肾上腺组织中克隆了恒河猴的cDNA，并使用外显子捕获策略从基因组DNA中克隆了黑猩猩和狒狒的cDNA。使用用恒河猴、狒狒、黑猩猩或人类P450c17转化的酵母微粒体，我们测量了17α-羟化酶和17,20裂解酶活性的米氏常数和最大反应速度。人类和黑猩猩的酶仅在两个氨基酸上有所不同，狒狒和恒河猴的P450c17仅在一个残基上有所不同；人类/黑猩猩的酶与狒狒/恒河猴的酶相差25 - 27个残基（95%的同一性）。令人惊讶的是，酶活性的最大差异是狒狒中P450c17的17α-羟化酶活性显著增加，它与恒河猴的不同仅在于255位残基[狒狒中为精氨酸（Arg），恒河猴中为组氨酸（His）]。255位残基在人类和黑猩猩中也是Arg。野生型人类P450c17及其Arg255His突变体具有相似的17α-羟化酶活性，但Arg255Ala突变体的17α-羟化酶活性降低。这些数据表明Arg255对17α-羟化酶活性很重要，并表明高等灵长类动物中肾上腺初现的进化不是由P450c17序列的变化决定的。
