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.
Highlights
Zebrafish and human cytochrome P450 (P450) 27C1 enzymes have been shown to be retinoid 3,4-desaturases
MS profiling was done with human liver and skin samples, based on previous work showing P450 27C1 mRNA in the liver [23] and the demonstration of retinol desaturation activity in human skin epidermis [41]
We hypothesized that the 372-amino acid entry in the databases is an error due to misassignment of the methionine start site and did extensive proteomic analysis with a second skin sample to define the protein found in human skin
Summary
MS profiling was done with human liver and skin samples, based on previous work showing P450 27C1 mRNA in the liver [23] and the demonstration of retinol desaturation activity in human skin epidermis [41]. Initial LC-MS/MS of tryptic peptides from a human skin sample showed the presence of P450 27C1, with 51% sequence coverage in this 372-residue region (supplemental Table S1) (Fig. 3). Immunoblotting consistently showed the presence of two bands in all five human skin samples examined (Fig. 4A) but no detectable bands in any of the human liver or kidney samples, only background staining (Fig. 4, B and C) (the limit of detection was 0.1 pmol of P450 27C1). The estimated content varied from 11 to 14 pmol of P450 27C1
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