Ile115Leu mutation in the SRS1 region of an insect cytochrome P450 (CYP6B1) compromises substrate turnover via changes in a predicted product release channel.
CYP6B1 represents the principal cytochrome P450 monooxygenase responsible for metabolizing furanocoumarins in Papilio polyxenes, an insect that specializes on host plants containing these toxins. Investigations of the amino acids responsible for the efficient metabolism of these plant toxins has identified Ile115 as one that modulates the rate of furanocoumarin metabolism even though it is predicted to be positioned at the edge of the heme plane and outside substrate contact regions. In contrast to previous expression studies conducted under conditions of limiting P450 reductase showing that the Ile115-to-Leu replacement enhances turnover of xanthotoxin and other furanocoumarins, studies conducted at high P450 reductase indicate that the Ile115-to-Leu replacement reduces turnover of these substrates. Further analysis of substrate binding affinities, heme spin state and NADPH consumption rates indicate that, whereas the I115L replacement mutant displays higher substrate affinity and heme spin state than the wild-type CYP6B1 protein, it utilizes NADPH more slowly than the wild-type CYP6B1 protein at high P450 reductase levels. Molecular models developed for the wild-type CYP6B1 and mutant protein suggest that more constricted channels extending from the catalytic site in the I115L mutant to the P450 surface limit the rate of product release from this mutant catalytic site under conditions not limited by the rate of electron transfer from NADPH.
Wen
Zhimou
Z
Department of Cell and Structural Biology, University of Illinois, Urbana, IL 61801, USA.
Baudry
Jerome
J
Berenbaum
May R
MR
Schuler
Mary A
MA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics; D002080:Butterflies / Q000201:enzymology; D020134:Catalytic Domain / Q000235:genetics; D004579:Electron Transport; D011564:Furocoumarins / Q000737:chemistry / Q000378:metabolism*; D007532:Isoleucine / Q000235:genetics; D007930:Leucine / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D013379:Substrate Specificity
D011564:Furocoumarins; D007532:Isoleucine; D001189:Aryl Hydrocarbon Hydroxylases; C465261:cytochrome P-450 CYP6B1 (butterfly); D007930:Leucine