Engineering, expression, and purification of "soluble" human cytochrome P45017alpha and its functional characterization.
To engineer a "soluble" form of membrane-bound cytochrome P45017alpha (CYP17)--a key enzyme in steroid hormone biosynthesis--in the present work we have built a computer model of the tertiary structure of the hemeprotein, identified the surface hydrophobic amino acid residues, substituted these residues for more hydrophilic ones, and expressed and purified hydrophilized forms of CYP17. We have constructed and purified the following mutant forms of human CYP17: CYP17dH (CYP17 with deleted hydrophobic N-terminal sequence (Delta(23))) and CYP17mod (CYP17dH with substituted cluster of hydrophobic amino acid residues in the region of the FG-loop). Removal of the N-terminal sequence responsible for interaction with the membrane does not dramatically change the association of the protein with the membrane. However, CYP17mod containing hydrophilic FG-loop is mostly localized in the cytosolic fraction. Thus, in the present work we for the first time engineered a "soluble" form of the usually membrane-bound human CYP17 that is not bound to membrane. The expression degree of CYP17mod is approximately 900 nmol/liter of culture. The hemeprotein can be purified to apparent homogeneity without using detergents at any purification step. It is shown that replacement of hydrophobic amino acid residues in the FG-loop region does not change the metabolic profile during hydroxylation of steroids that is characteristic for wild type CYP17. Besides, the modification of the hemeprotein does not affect the affinity of CYP17 to steroid substrates. The engineered "soluble" form of human CYP17 is used as a subject for crystallization of the hemeprotein.
Pechurskaya
T A
TA
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, 220141, Belarus.
Lukashevich
O P
OP
Gilep
A A
AA
Usanov
S A
SA
D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013254:Steroid 17-alpha-Hydroxylase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D013254:Steroid 17-alpha-Hydroxylase