Enhanced electron transfer and lauric acid hydroxylation by site-directed mutagenesis of CYP119.
CYP119, a cytochrome P450 from a thermophilic organism for which a crystal structure is available, is shown here to hydroxylate lauric acid in a reaction supported by putidaredoxin and putidaredoxin reductase. This fatty acid hydroxylation activity is increased 15-fold by T214V and D77R mutations. The T214V mutation increases the rate by facilitating substrate binding and enhancing the associated spin state change, whereas the D77R mutation improves binding of the heterologous redox partner putidaredoxin to CYP119 and the rate of electron transfer from it to the heme group. A sequence alignment with P450(cam) can, therefore, be used to identify a part of the binding site for putidaredoxin on an unrelated P450 enzyme. This information can be used to engineer by mutagenesis an improved complementarity of the protein-protein interface that results in improved electron transfer from putidaredoxin to the P450 enzyme. As a result, the catalytic activity of the thermo- and barostable CYP119 has been incorporated into a catalytic system that hydroxylates fatty acids.
Koo
Laura S
LS
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446, USA.
Immoos
Chad E
CE
Cohen
Michael S
MS
Farmer
Patrick J
PJ
Ortiz de Montellano
Paul R
PR
D000595:Amino Acid Sequence; D019843:Archaeal Proteins; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004852:Epoxy Compounds / Q000737:chemistry / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D007850:Lauric Acids / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D020058:Styrene / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D019843:Archaeal Proteins; D004852:Epoxy Compounds; D005288:Ferredoxins; D007850:Lauric Acids; C030358:lauric acid; D020058:Styrene; C100137:putidaredoxin; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C408874:CYP119 protein, Sulfolobus solfataricus; D009247:NADH, NADPH Oxidoreductases; C022486:putidaredoxin reductase