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J Biol Chem. 2017 Aug 18;292(33):13672-13687. doi: 10.1074/jbc.M116.773937. Epub 2017 Jul 12.

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

Author information

1
From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
2
From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 f.guengerich@vanderbilt.edu.

Abstract

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-trans-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 kcat/Km ) 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.

KEYWORDS:

cytochrome P450; enzyme kinetics; isotope effect; mass spectrometry (MS); pre-steady-state kinetics; retinol; skin

PMID:
28701464
PMCID:
PMC5566523
DOI:
10.1074/jbc.M116.773937
[Indexed for MEDLINE]
Free PMC Article

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