Alteration of the substrate specificity of mouse 2A P450s by the identity of residue-209: steroid-binding site and orientation.
Mouse steroid 7α- and 15α-hydroxylases (P450c7 and P450c15) and coumarin 7-hydroxylase (P450coh) are structurally similar. To study the structural basis of the substrate specificities of these enzymes, we constructed a series of the mutant P450s, expressed in COS-1 and yeast cells, and studied them spectroscopically as well as enzyme-kinetically. A single amino acid mutation of residue-209 is sufficient to alter the substrate specificity of the P450s from xenobiotics to steroids and subsequently, from testosterone to corticosterone. Moreover, residue-209, when it is asparagine, appears to bind directly to the 11β-hydroxyl of corticosterone. The mutations also after the spin equilibrium of P450 depending on the hydrophobicity and size of residue-209. We conclude, therefore, that residue-209 resides close to the 6th ligand of heme in the mouse 2A subfamily and is located at a critical site of the substrate-binding pocket. As a result, the identity of the residue-209 plays a key role in determining the substrate specificity.
Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
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M
Negishi
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M
Iwasaki
R O
RO
Juvonen
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K
Aida
D019943:Amino Acid Substitution; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002790:Cholesterol 7-alpha-Hydroxylase; D065637:Cytochrome P-450 CYP2A6; D051379:Mice; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D050505:Mutant Proteins; D011994:Recombinant Proteins; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; C050356:steroid 15-alpha-hydroxylase; C029009:steroid hormone 7-alpha-hydroxylase; D002790:Cholesterol 7-alpha-Hydroxylase