Human mitochondrial cytochrome P450 27C1 is localized in skin and preferentially desaturates -retinol to 3,4-dehydroretinol.

Johnson KM, Phan TTN, Albertolle ME, Guengerich FP
J Biol Chem. 2017 292 (33): 13672-13687

PMID: 28701464 · PMCID: PMC5566523 · DOI:10.1074/jbc.M116.773937

Recently, zebrafish and human cytochrome P450 (P450) 27C1 enzymes have been shown to be retinoid 3,4-desaturases. The enzyme is unusual among mammalian P450s in that the predominant oxidation is a desaturation and in that hydroxylation represents only a minor pathway. We show by proteomic analysis that P450 27C1 is localized to human skin, with two proteins of different sizes present, one being a cleavage product of the full-length form. P450 27C1 oxidized all--retinol to 3,4-dehydroretinol, 4-hydroxy (OH) retinol, and 3-OH retinol in a 100:3:2 ratio. Neither 3-OH nor 4-OH retinol was an intermediate in desaturation. No kinetic burst was observed in the steady state; neither the rate of substrate binding nor product release was rate-limiting. Ferric P450 27C1 reduction by adrenodoxin was 3-fold faster in the presence of the substrate and was ∼5-fold faster than the overall turnover. Kinetic isotope effects of 1.5-2.3 (on / ) were observed with 3,3-, 4,4-, and 3,3,4,4-deuterated retinol. Deuteration at C-4 produced a 4-fold increase in 3-hydroxylation due to metabolic switching, with no observable effect on 4-hydroxylation. Deuteration at C-3 produced a strong kinetic isotope effect for 3-hydroxylation but not 4-hydroxylation. Analysis of the products of deuterated retinol showed a lack of scrambling of a putative allylic radical at C-3 and C-4. We conclude that the most likely catalytic mechanism begins with abstraction of a hydrogen atom from C-4 (or possibly C-3) initiating the desaturation pathway, followed by a sequential abstraction of a hydrogen atom or proton-coupled electron transfer. Adrenodoxin reduction and hydrogen abstraction both contribute to rate limitation.

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

MeSH Terms (20)

Biocatalysis Cytochrome P450 Family 27 Gene Expression Profiling Gene Expression Regulation, Enzymologic Humans Hydrogenation Hydroxylation Isoenzymes Kinetics Mitochondria Molecular Structure Organ Specificity Oxidation-Reduction Peptide Fragments Proteolysis Proteomics Skin Stereoisomerism Substrate Specificity Vitamin A

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