Identification by in Vitro Mutagenesis of the Interaction of Two Segments of C2MstC1, a Chimera of Cytochromes P450 2C2 and P450 2C1

Manjunath K Ramarao,Petra Straub,Byron Kemper

doi:10.1074/jbc.270.4.1873

Abstract

A hybrid cytochrome P450, C2MstC1, with 306 N-terminal amino acids derived from cytochrome P450 2C2 sequence and 184 C-terminal amino acids from cytochrome P450 2C1 acquires a novel progesterone 21-hydroxylase activity which is absent in the parent enzymes. Extension of the cytochrome P450 2C2 sequence to residue 382 reduced progesterone hydroxylase activity to 5% of that of C2MstC1, while further extension to residue 411 or 462 increased activity back to about 30 or 40%, respectively. In the chimera with cytochrome P450 2C2 sequence to residue 382, substitution of cytochrome P450 2C1 amino acids at positions 368, 369, and 374 increased progesterone hydroxylase activity to a level equivalent to that of C2MstC1. In the chimera with cytochrome P450 2C2 sequence extending to residue 411, substitutions of P450 2C1 amino acids at positions 386 and 388, in addition those at 368, 369, and 374, were required to obtain activities equivalent to that of C2MstC1, which suggests an interaction between these two regions. The lauric acid hydroxylase activities of all chimeras and mutant cytochromes P450 differed by 2-fold or less, demonstrating that the changes in progesterone hydroxylase activity reflected altered interactions with the substrate. Alignment of cytochrome P450 2C1 sequence with cytochromes P450cam, P450BM-3, and P450terp predicts that residues 368/369 and 386/388 are in adjacent antiparallel strands of the same beta-sheet, in agreement with the experimental data suggesting an interaction between these two regions.

Highlights

A hybrid cytochrome P450, C2MstCI, with 306 N-terminal amino acids derived from cytochrome P450 2C2 sequence and 184 C-terminal amino acids from cytochrome P450 2CI acquires a novel progesterone 21-hydroxylase activity which is absent in the parent enzymes
In the chimera with cytochrome P450 2C2 sequence extending to residue 411, substitutions of P450 2Cl amino acids at positions 386 and 388, in addition those at [368, 369], and 374, were required to obtain activities equivalent to that of C2MstCI, which suggests an interaction between these two regions
To initially identify the specific P450 2Cl C-terminal sequence required for progesterone 2l-hydroxylase activity, additional chimeric P450s were constructed with successively shorter C-terminal sequences from P450 2Cl (Fig. 1)

Summary

The abbreviations used are

P450, cytochrome P450; HPLC, high pressure liquid chromatography, PAGE, polyacrylamide gel electrophoresis; SRS, substrate recognition site; PCR, polymerase chain reaction. We have shown that the amino acids in the N-tenninal portion ofP450 2C2 are important for the progesterone hydroxylase activity of the chimera and for the lauric acid hydroxylase activity of both the chimera and P450 2C2 (5, 7, 8) These amino acids are within SRS-l, one of six such sites predicted from the alignment of P450 2C sequences with P450cam , for which the three-dimensional structure is known (9). The acquisition of progesterone hydroxylase activity when the C-terminal 184 amino acids of P450 2Cl were substituted for those of P450 2C2 indicates that regions in the C-tenninal portion of the P450 are critical for recognition of steroid substrates. These experimental data are consistent with the relative positions of these regions predicted by alignment with the amino acid sequences of bacterial P450s for which three-dimensional structures have been determined

EXPERIMENTAL PROCEDURES

RESULTS

Sty C1 Mutants