Precursor of ether phospholipids is synthesized by a flavoenzyme through covalent catalysis

Abstract

The precursor of the essential ether phospholipids is synthesized by a peroxisomal enzyme that uses a flavin cofactor to catalyze a reaction that does not alter the redox state of the substrates. The enzyme crystal structure reveals a V-shaped active site with a narrow constriction in front of the prosthetic group. Mutations causing inborn ether phospholipid deficiency, a very severe genetic disease, target residues that are part of the catalytic center. Biochemical analysis using substrate and flavin analogs, absorbance spectroscopy, mutagenesis, and mass spectrometry provide compelling evidence supporting an unusual mechanism of covalent catalysis. The flavin functions as a chemical trap that promotes exchange of an acyl with an alkyl group, generating the characteristic ether bond. Structural comparisons show that the covalent versus noncovalent mechanistic distinction in flavoenzyme catalysis and evolution relies on subtle factors rather than on gross modifications of the cofactor environment.

Discovery of Inhibitors for the Ether Lipid-Generating Enzyme AGPS as Anti-Cancer Agents

“…While the precise roles of intracellular and circulating ether lipids is not yet clear, their particular physicochemical properties contribute to their biological importance in cellular structure, membrane fusion and vesicle formation, free radicals scavenging, storage of lipid second messengers, and lipid signaling molecules. Ether lipid synthesis occurs in peroxisomes and begins with the esterification of dihydroxyacetone phosphate (DHAP) with a long-chain fatty acyl-CoA ester by the enzyme DHAP acyl-transferase (DHAPAT) and subsequent replacement of the fatty acyl chain by a fatty alcohol to form alkyl-DHAP by alkyl-glycerone phosphate synthase (AGPS) (Figure 1) [6–8]. …”

Recombinant human dihydroxyacetonephosphate acyl-transferase characterization as an integral monotopic membrane protein

“…Purified recombinant ADPS (1 μM), prepared according to Nenci et al., 2012 [11], and the radioactive substrate (100 μM), [1–14C]hexadecyl-DHAP (Sigma-Aldrich; specific radioactivity adjusted to 13,000 dpm/nmol), were added to the protein solution after the incubation. Once ADPS was added, small aliquots (10 μl) were taken at 1.5 min intervals and deposited on a DEAE cellulose membrane (Whatman), which interact with the phosphate groups of DHAP.…”

“…All the assays were performed in duplicate. The positive control was performed as described above, using ADPS (1 μM) and adding 100 μM palmitoyl-DHAP [11].…”

“…retaining the His 8 -tag). Purified ADPS was prepared according to Nenci et al., 2012 [11]. The two proteins were mixed with nickel-resin, equilibrated with buffer containing 50 mM bicine pH 8.2, 50 mM NaCl, 5% glycerol and 0.05% FSC-12.…”

“…Whereas the unusual reaction mechanism of ADPS flavin fueled the interest in the biochemical characterization of this enzyme [10], [11], [12], the difficulties associated to DHAPAT expression and purification slowed progress for decades. DHAPAT performs the first step of the ether phospholipid biosynthetic pathway, namely the acylation of DHAP to form acyl-DHAP, which is the substrate of the downstream ADPS reaction.…”