Expression and purification of functional human 17 alpha-hydroxylase/17,20-lyase (P450c17) in Escherichia coli. Use of this system for study of a novel form of combined 17 alpha-hydroxylase/17,20-lyase deficiency.
Enzymatically active human 17 alpha-hydroxylase cytochrome P450 (P450c17) has been expressed in and purified from Escherichia coli. The cDNA containing modifications within the amino-terminal eight codons which are favorable for expression in E. coli, as well as codons for 4 histidine residues at the carboxyl terminus, was placed in the pCWori+ expression vector. The modified human P450c17 was detected spectrophotometrically (400 nmol of P450c17/liter culture) and was found to be integrated into E. coli membranes. This previously inaccessible human P450 was purified to electrophoretic homogeneity (10.7 nmol of P450/mg) from solubilized bacterial membranes using two sequential chromatographic steps, nickel nitrilotriacetate followed by hydroxylapatite. The expected enzymatic activities of human P450c17 were reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase, giving turnover numbers of 8.0 nmol/min/nmol P450 for pregnenolone, 6.5 nmol/min/nmol P450 for progesterone, 0.06 nmol/min/nmol P450 for 17 alpha-hydroxypregnenolone, and no detectable activity for 17 alpha-hydroxyprogesterone. This system was utilized to study the molecular basis of a novel form of combined 17 alpha-hydroxylase, 17,20-lyase deficiency resulting from compound heterozygous mutations, a missense point mutation Tyr64(TAT)--> Ser (TCT), and an Ile112 duplication (ATCATC). Upon expression of these mutant proteins in E. coli, the Tyr64 mutant has 15% of the wild type 17 alpha-hydroxylase activity, whereas the Ile112 duplication shows no activity, results consistent with the observed clinical phenotype.
Department of Biochemistry and Obstetrics, University of Texas Southwestern Medical Center, Dallas 75235.
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T
Imai
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H
Globerman
J M
JM
Gertner
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N
Kagawa
M R
MR
Waterman
D000324:Adrenocorticotropic Hormone; D000595:Amino Acid Sequence; D000728:Androgens / Q000097:blood; D000818:Animals; D001483:Base Sequence; D006063:Chorionic Gonadotropin; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007223:Infant; D008099:Liver / Q000201:enzymology; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010957:Plasmids; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine; D013254:Steroid 17-alpha-Hydroxylase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D013256:Steroids / Q000097:blood / Q000652:urine; D013737:Testis / Q000201:enzymology; D014443:Tyrosine
D000728:Androgens; D006063:Chorionic Gonadotropin; D011994:Recombinant Proteins; D013256:Steroids; D014443:Tyrosine; D012694:Serine; D000324:Adrenocorticotropic Hormone; D004247:DNA; D013254:Steroid 17-alpha-Hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase