Construction and expression of human/bovine P45017 alpha chimeric proteins: evidence for distinct tertiary structures in the same P450 from two different species.
In the human and bovine adrenal cortex, 17 alpha-hydroxylase (P45017 alpha) catalyzes reactions involved in the production of C21-glucocorticoids (17 alpha-hydroxylation) and C19-androgens (17,20-lyase). The bovine and human forms of P45017 alpha share 71% primary sequence identity. Using naturally occurring restriction sites common to cDNAs encoding both human and bovine P45017 alpha, we have constructed bovine/human (bovine amino terminus and human carboxy terminus) and human/bovine (human amino terminus and bovine carboxy terminus) cDNAs that have been expressed in COS 1 cells, and the enzymatic properties of the resultant chimeric proteins have been examined. The three bovine/human chimeras studied have 17 alpha-hydroxylase activities intermediate between those of the wild-type bovine and wild-type human enzymes, although the 17,20-lyase activity of these chimeras is significantly lower than that of either of the wild-type enzymes. Surprisingly, the opposite chimeras (those containing a human amino-terminal sequene and a bovine carboxy-terminal sequence) are all virtually inactive, even though they appear to be expressed at normal levels. These results indicate that the folding of P45017 alpha initiated by the bovine amino terminus can accommodate human P45017 alpha sequences of various lengths to produce a relatively normal 17 alpha-hydroxylase having decreased 17,20-lyase activity. On the other hand, folding initiated by the human P45017 alpha amino terminus does not easily accommodate bovine carboxy-terminal sequences to produce a functional enzyme. Presumably this difference arises from the fact that the tertiary structures of the bovine and human forms of P45017 alpha are sufficiently different so that interchanging sequences will not lead to functional enzymes in a predictable fashion.
Lorence
M C
MC
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Trant
J M
JM
Clark
B J
BJ
Khyatt
B
B
Mason
J I
JI
Estabrook
R W
RW
Waterman
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004247:DNA / Q000235:genetics; D005813:Genes, Synthetic; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D004247:DNA; D013254:Steroid 17-alpha-Hydroxylase