Site-directed mutagenesis of cytochrome P450eryF: implications for substrate oxidation, cooperativity, and topology of the active site.
The role of five active-site residues (Phe-78, Gly-91, Ser-171, Ile-174, and Leu-175) has been investigated in P450eryF, the only bacterial P450 known to show cooperativity. The residues were selected based on two-ligand-bound P450eryF structures and previous mutagenesis studies of other cytochromes P450. To better understand the role of these residues in substrate catalysis and cooperativity, each mutant was generated in the wild-type and A245T background, a substitution that enables P450eryF to oxidize testosterone and 7-benzyloxyquinoline (7-BQ). Replacement of Phe-78 with tryptophan decreased cooperativity of 9-aminophenanthrene binding, with little effect on testosterone binding or oxidation. Interestingly, substitution of Gly-91 with alanine or phenylalanine abolished the type-I spectral change elicited by testosterone and significantly decreased testosterone hydroxylation. However, G91A/A245T showed a 4-fold higher k(cat) value with 7-BQ compared with A245T. Replacement of Ser-171 with alanine or phenylalanine did not alter cooperativity of testosterone binding but significantly decreased binding affinity and oxidation of testosterone and 7-BQ. The only mutant that exhibited an increased testosterone binding affinity and increased rates of testosterone and 7-BQ oxidation was I174F. Substitution of Ile-175 with phenylalanine decreased testosterone and 7-BQ oxidation. Reaction with phenyldiazene showed that P450eryF may be much more open above pyrrole ring B than other cytochromes P450 and indicated significant changes in active-site topology in some of the mutants. The study suggests a crucial role of residues Ser-171, Ile-174, and Leu-175, which are part of a distal ligand site, in addition to the proximal Gly-91 in determining the oxidative properties of P450eryF.
Khan
Kishore K
KK
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Boulevard, Galveston 77555-1031, USA. kkkhan@utmb.edu
He
You Ai
YA
He
You Qun
YQ
Halpert
James R
JR
D001426:Bacterial Proteins; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011524:Protoporphyrins / Q000378:metabolism; D011804:Quinolines / Q000378:metabolism; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D001426:Bacterial Proteins; D008024:Ligands; D011524:Protoporphyrins; D011804:Quinolines; C065956:7-benzyloxyquinoline; D013739:Testosterone; C032609:N-phenylprotoporphyrin IX; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea