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-desaturated intermediates in ergosterol (fungi), phytosterol (plants), and cholesterol (animals) biosynthesis. During the catalytic cycle, a substrate undergoes three successive monooxygenation reactions resulting in formation of 14-hydroxymethyl, 14-carboxaldehyde, and 14-formyl derivatives followed by elimination of formic acid with introduction of a 14,15 double bond (1, 2).Being a key enzyme of sterol biosynthesis, CYP51 has been a target for antifungal (3) and cholesterol-lowering (4) drug design. The first generation of antifungal inhibitors of CYP51, fluconazole (FLU) and itraconazole, have revolutionized treatment of some serious fungal infections. However, the treatment of others is still far from satisfactory, and there is a need for new broad-and narrow-spectrum antifungal agents (3). Furthermore, fungal resistance caused by acquisition of intrinsically resistant species, e.g., Aspergillus fumigatus, or by mutation of initially susceptible strains, e.g., Candida albicans, is an increasing clinical problem (5, 6) forcing the development of new triazole antifungals. Azole antifungals selectively inhibit yeast and fungal CYP51 over their plant and human counterparts (7), but crossover inhibition of CYP51 in two different species can cause undesirable side effects and is another reason for the continuing search for better agents (3,8). The problem of specificity is being addressed empirically by exploring inhibitors of different structures and by efforts to develop threedimensional molecular models of CYP51-active sites based on primary sequence analyses and available structures for bacterial P450s. These models initially were based on the structure of P450cam (9, 10) and more recently on the structure of P450BM3 (11-14) because of its higher sequence similarity.The available P450 structures show that the overall P450 structural fold is preserved during evolution from bacteria through mammals (15)(16)(17)(18)(19)(20)(21)(22). At the same time, there are variable regions that appear to be associated with recognition and binding of structurally diverse substrates and redox partners (23). Experimental structural information on the active sites of the fungal, plant, and mammalian CYP51 would greatly facilitate developing more efficacious antifungal drugs. However, until recently all known forms of CYP51 were membrane-bound microsomal enzymes, which complicated structural studies of this protein by x-ray crystallography. A soluble CYP51 ortholog discovered recently in Mycobacterium tuberculosis (24) exhibits 35-38% sequence identity to plant, 33-35% to animal, and 26-29% to fungal enzymes. Although MTCYP51 can oxidize lanosterol and 24,25-dihydrolanosterol in vitro, the plant substrate obtusifoliol is preferred (25). We have crystallized Escherichia coli-expressed MTCYP51 in the presence of two different azole inhibitors, 4-phenylimidazole (4-PI) and FLU, and report here their structures at 2.1 and 2.2 Å, respectively. Materials and MethodsMTCYP51 was expressed and purified as described (25). Protein of appro...

When the cDNA encoding bovine microsomal 17a-hydroxylase cytochrome P450 (P45017a) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017a per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017a can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017a enzyme. Surprisingly, it is found that E. coli contain an electrontransport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17a-hydroxylase and 17,20-lyase activities of P45017a. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structurefunction analysis of the cytochrome P450 system. Microsomal cytochromes P450 are integral membrane hemoproteins that catalyze the oxidative metabolism of a wide variety of endogenous and exogenous compounds. Deriving reducing equivalents from NADPH via a membrane-bound flavoprotein oxidoreductase (NADPH-cytochrome P450 reductase), these mixed-function oxidases activate molecular oxygen so as to insert one atom into a lipophilic substrate and the other atom into water. Recent study of the molecular aspects underlying eukaryotic cytochrome P450 structure and function has relied on the techniques of molecular biology to synthesize specific individual forms ofcytochrome P450 in heterologous expression systems. Yeast (1), COS 1 (2), and eukaryotic cells infected with a viral vector (3, 4) have been used as hosts for the heterologous expression of cytochrome P450 molecules; however, each has limitations to their usefulness as systems for structure-function analysis. Although the bacterium Escherichia coli has demonstrated great usefulness in the expression of many prokaryotic and eukaryotic proteins, E. coli as an expression system for cytochrome P450 has been limited primarily to the soluble prokaryotic forms of this gene superfamily (5). We have used the cDNA encoding bovine 17a-hydroxylase cytochrome P450 (P45017a) to examine the utility of E. coli as an expression system for eukaryotic cytochromes P450 in the hopes that such a system might prove suitable for both enzymatic and structural studies. This microsomal cytochrome P450 catalyzes the regiospecific and stereospecific 17a-hydroxylation of the C21 steroids pregnenolone and progesterone in the pathway leading to the production of cortisol and the 17,20-lyase conversion of 17a-hydroxypregnenolone to the C19 adrenal androgen dehydroepiandrosterone (DHEA) in the adrenal cortex of most mammalian species. P45017a also converts these 17a-hydroxylat...

SummaryThe CYP51 family is an intriguing subject for fundamental P450 structure/function studies and is also an important clinical drug target. This review updates information on the variety of the CYP51 family members, including their physiological roles, natural substrates and substrate preferences, and catalytic properties in vitro. We present experimental support for the notion that specific conserved regions in the P450 sequences represent a CYP51 signature. Two possible roles of CYP51 in P450 evolution are discussed and the major approaches for CYP51 inhibition are summarized.Keywords sterol 14α-demethylase (CYP51); sterol biosynthesis; substrate preferences; catalytic activity; inhibition Family overviewSterol 14α-demethylation as a general part of sterol biosynthetic pathways in eukaryotes [1] has been known and studied for more than 30 years [2][3][4][5][6][7]. The enzyme catalyzing this reaction was first purified from yeast in 1984 (Sacharomyces cerevisiea [8]), and following determination of its primary structure [9] the cytochrome P450 sterol 14α-demethylases were placed into the CYP51 family, a number reserved for fungal sequences [10]. In 1986 the orthologous mammalian P450 was purified from rat liver microsomes [11], in 1996 the first sterol 14α-demethylase was found in plants (Sorghum bicolor [12]), and in 2000 the orthologous nature of a CYP51-like gene [13] from Mycobacterium tuberculosis to eukaryotic CYP51s was confirmed [14].Currently the CYP51 family joins proteins found in 82 organisms from all biological kingdoms. In addition, several plants and fungi contain multiple CYP51 genes (e.g. rice (10), black oats (2), tobacco (2), Arabidopsis thaliana (2), Fuzarium graminearum (3) or Aspergillus species: A. fumigatus (2), A.nidulans (2), A. orizae (3)). As a result, the number of known CYP51 sequences exceeds 100. The reasons for the existence of homologous CYP51 genes in the same species or their precise functions remain unknown, though it was reported that only one of the two CYP51 genes from A. thaliana (CYP51A2) is functional, while the other is an expressed pseudogene [15]. Mammalian genomes contain only one CYP51 gene yet sometimes † Corresponding author: Michael R. Waterman, Tel.: 615-343-1373; Fax: 615-322-4349; E-mail: michael.waterman@vanderbilt.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The average amino acid sequence identity in the CYP51 family is about 30%, varying from relatively high between the proteins from evolutionary closely related species (e.g. 95% in mammals) to lower values within the highly diverse kingdoms of lower...