Yeast Mpo1 Is a Novel Dioxygenase That Catalyzes the α-Oxidation of a 2-Hydroxy Fatty Acid in an Fe²⁺-Dependent Manner

Abstract

Phytosphingosine (PHS) is the major long-chain base component of sphingolipids in Saccharomyces cerevisiae. The PHS metabolic pathway includes a fatty acid (FA) α-oxidation reaction. Recently, we identified the novel protein Mpo1, which is involved in PHS metabolism. However, the details of the FA α-oxidation reaction and the role of Mpo1 in PHS metabolism remained unclear. In the present study, we revealed that Mpo1 is involved in the α-oxidation of 2-hydroxy (2-OH) palmitic acid (C16:0-COOH) in the PHS metabolic pathway. Our in vitro assay revealed that not only the Mpo1-containing membrane fraction but also the soluble fraction was required for the α-oxidation of 2-OH C16:0-COOH. The addition of Fe2+ eliminated the need for the soluble fraction. Purified Mpo1 converted 2-OH C16:0-COOH to C15:0-COOH in the presence of Fe2+, indicating that Mpo1 is the enzyme body responsible for catalyzing the FA α-oxidation reaction. This reaction was also found to require an oxygen molecule. Our findings indicate that Mpo1 catalyzes the FA α-oxidation reaction as 2-OH fatty acid dioxygenase, mediated by iron(IV) peroxide. Although numerous Mpo1 homologs exist in bacteria, fungi, protozoa, and plants, their functions had not yet been clarified. However, our findings suggest that these family members function as dioxygenases.

The lipid composition of yeast cells modulates the response to iron deficiency

“…Therefore, iron depletion alters the sphingolipid synthesis pathway [20]. Moreover, a recent report has shown that Mpo1 is a novel iron-dependent dioxygenase involved in phytosphingosine metabolism [49,50]. Various studies suggest that sphingolipid metabolism could also modulate Aft1 function.…”

Catalytic residues, substrate specificity, and role in carbon starvation of the 2-hydroxy FA dioxygenase Mpo1 in yeast

“…The -oxidation reactions differ between mammals and yeast. Mammalian -oxidation involves three steps (CoA addition, long-chain aldehyde production by cleavage between the C1 and C2 positions, and oxidation to FAs), whereas yeast -oxidation is conducted in one step (17,18,24). Accordingly, the proteins involved in -oxidation differ between mammals and yeast.…”

“…We previously identified MPO1 as the gene functioning in the degradation pathway of PHS in yeast (17). It subsequently became clear that Mpo1 is involved in the oxidation of 2-OH FAs as an Fe 2+ -dependent dioxygenase (EC 1.14.18.12), which directly converts 2-OH LCFAs to nonhydroxy FAs with one less carbon (24).…”

“…However, there remain many unsolved problems regarding the structural and enzymatic characterization of Mpo1. For instance, it is an ER protein that is predicted to have some transmembrane domains, but the exact number of these domains and its membrane topology are unknown; the Mpo1-catalyzed -oxidation reaction requires Fe 2+ , but the region and amino acid residues involved in Fe 2+ binding are unclear; and Mpo1 exhibits activity toward 2-OH LCFAs derived from PHS (24), but it is unclear whether Mpo1 is involved in -oxidation of 2-OH VLCFAs derived from the FA portion of sphingolipids. Although the 2-OH C26 VLCFA is abundant in yeast sphingolipids, its degradation process is unclear, including whether it is subject to -oxidation by Mpo1.…”

A comprehensive mechanistic model of iron metabolism inSaccharomyces cerevisiae

“…Mpo1 is an Fe II -containing dioxygenase in the ER membrane that catalyzes the oxidation of a 2-hydroxy fatty acid. 190 Unlike the other biosynthetic enzymes just discussed, Mpo1 helps degrade a membrane component rather than synthesize one.…”