Identification by site-directed mutagenesis of two lysine residues in cholesterol side chain cleavage cytochrome P450 that are essential for adrenodoxin binding

A Wada,M.R Waterman

doi:10.1016/s0021-9258(18)50028-4

Abstract

Utilizing site-directed mutagenesis and an Escherichia coli expression system for bovine cholesterol side chain cleavage cytochrome P450, lysine residues at 377 and 381 are found to play crucial roles in binding bovine adrenodoxin, required for transfer of electrons to mitochondrial P450s. These lysine residues are conserved among mitochondrial P450s and have been implicated previously by chemical modification studies as being important for adrenodoxin binding. In the present study, site-directed mutagenesis producing either neutral or positive amino acids at 377 or 381 has no effect on the structure of side chain cleavage cytochrome P450 as determined spectrally or on the enzymatic conversion of cholesterol to pregnenolone. However, the estimated Ks of adrenodoxin binding is increased approximately 150-600-fold depending on the particular mutation. Therefore these conserved positively charged residues in mitochondrial P450s are the key sites for adrenodoxin binding which is electrostatic in nature.

Highlights

374 PLLKASIKETLRLH 387 richia coli expression systemforbovinecholesterol side chain cleavage cytochrome P450, lysine residues at 377 and 381 are foundtoplaycrucial roles in binding bovine adrenodoxin, required for transfer of
Charged residues in mitochondrial P450s are the key sites for adrenodoxin binding whichis electrostatic in Alignment of amino acid sequences of mitochondrial P450s nature
The interaction of P450, and adrenodoxin has been reported to be electrostatic [2], and recently Coghlan and Vickery [3] determined by site-directed mutagenesis that asparticacid residues at positions 76 and 79 of human adrenodoxin are important for binding to P45OS

Summary

RESULTS

E. coli is located in the membrane fraction of the bacteria in position 328 instead of AAC (Asn) aspreviously reported [5]. These expressed in E. coli is low spin andshows the expected spectral partially purified, mutated forms ofP450,,, havea specific change to the high spin form by the addition of substrate content of 1-2 nmol P450/mg protein after hydroxyapatite [28]. Upon addition acids at 377 and 381 might have on the enzymatic activity of of much larger amounts of adrenodoxin were spectral changes cholesterol side chain cleavage, we measured pregnenolone induced in mutated P450.,, (Fig. 5B). Enzymatic activity of the cholesterol side chain cleavage rection of wild type and mutated forms of P450, with different concentrations of adrenodoxin

Wild type

DISCUSSION

Enzymatic activity was also observed with mutant forms of

Study of the single mutants showed that at least the basic