Amino acids responsible for the novel progesterone C-21 hydroxylase activity in a chimera of cytochrome P-450 2C2/2C1
Ramarao, Manjunath K.
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https://hdl.handle.net/2142/20602
Description
Title
Amino acids responsible for the novel progesterone C-21 hydroxylase activity in a chimera of cytochrome P-450 2C2/2C1
Author(s)
Ramarao, Manjunath K.
Issue Date
1994
Doctoral Committee Chair(s)
Kemper, Byron W.
Department of Study
Molecular and Integrative Physiology
Discipline
Physiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Cell
Biology, Animal Physiology
Language
eng
Abstract
A hybrid cytochrome P450, C2MstC1, with 306 amino terminal P450 2C2 amino acids and 184 carboxy-terminal P450 2C1 amino acids acquires a novel progesterone C-21 hydroxylase activity which is absent in the parent enzymes. Extension of P450 2C2 sequence to residue 383 (C2StyC1), reduced progesterone hydroxylase activity to 5% of that of C2MstC1, while further extension to residue 411 (C2BalC1), increased activity back to about 30%. Only 28 carboxy-terminal amino acids from P450 2C1 (C2HincC1) were required for activity 40% of that of C2MstC1. In C2StyC1, substitution of P450 2C1 amino acids at positions 368, 369, and 374 increased progesterone hydroxylase activity greater than that in C2MstC1. In C2BalC1 however, additional substitutions of P450 2C1 amino acids at positions 386 and 388 were required to obtain activities greater than that of C2MstC1. These results suggest that an interaction occurs between residues 368, 369 or 374 and residues 386 or 388 which affects the binding of progesterone or its accessibility to the active site of the enzyme. These five changes confer progesterone hydroxylase activity to P450 2C2 but only about 10% of that of C2MstC1. Mutational analysis of the 28 carboxy-terminal amino acids of P450 2C2 revealed that valine 473 was an additional requirement for an increase in the progesterone C-21 hydroxylase activity, comparable to that of C2MstC1. In contrast to the progesterone hydroxylase activity, the lauric acid hydroxylase activities of all chimeras and mutant cytochromes P450 differed by 2-fold or less demonstrating that the changes in progesterone activities reflected altered interactions with the substrate rather than general effects on enzyme activity or expression of functional protein. An exception was the valine 477 glycine mutation, in which activity for both substrates was reduced and the regiospecificity of hydroxylation was altered. Molecular modeling of the substrate binding pockets of P450 2C2 and its mutant consisting of P450 2C1 residues at the above mentioned positions, based on the three dimensional structure of the bacterial cytochrome P450cam suggest that the P450 2C1 amino acids facilitate progesterone binding by providing a larger, more hydrophobic substrate binding pocket and/or through interactions with the heme.
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