Stereochemistry of a bifunctional dihydroceramide delta 4-desaturase/hydroxylase from Candida albicans; a key enzyme of sphingolipid metabolism.
The stereochemical course of the dihydroceramide delta 4-(E)-desaturase from Candida albicans, cloned and expressed in the yeast Saccharomyces cerevisiae strain sur2 delta, was determined using stereospecifically labelled (2R,3S)-[2,3,4,4-2H4]-palmitic acid as a metabolic probe. Mass spectrometric analysis of the dinitrophenyl-derivatives of the labelled long-chain bases revealed elimination of a single deuterium atom from C(4) (corresponding to the C(4)-HR) along with a hydrogen atom from C(5) (corresponding to the C(5)-HS). This finding is consistent with an overall syn-elimination of the two vicinal hydrogen atoms. Besides the desaturation product sphingosine (93%) minor amounts of a 4-hydroxylated product (phytosphinganine, 7%) were identified that classify the Candida enzyme as a bifunctional desaturase/hydroxylase. Both processes, desaturation and hydroxylation proceed with loss of C(4)-HR from the chiral precursor. This finding is in agreement with a two-step process involving activation of the substrate by removal of the C(4)-HR to give a C-centred radical or radicaloid followed by either disproportionation into an olefin, water and a reduced diiron complex, or to recombination of the primary reactive intermediate with an active site-bound oxygen to yield a secondary alcohol. This result demonstrates the close mechanistic relationship between desaturation and hydroxylation as two different reaction pathways of a single enzyme and strengthens the mechanistic relationship of desaturases from fatty acid metabolism and sphingolipids.
Beckmann
Christoph
C
Max-Planck-Institut für Chemische Okologie, Bioorganische Chemie, Winzerlaer Str. 10, D-07745 Jena, Germany.
Rattke
Janine
J
Sperling
Petra
P
Heinz
Ernst
E
Boland
Wilhelm
W
D002176:Candida albicans / Q000201:enzymology*; D004140:Dinitrophenols / Q000737:chemistry / Q000378:metabolism; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D006900:Hydroxylation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019308:Palmitic Acid / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013107:Sphingolipids / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D013237:Stereoisomerism; D014316:Tritium
D004140:Dinitrophenols; D011994:Recombinant Proteins; D013107:Sphingolipids; D014316:Tritium; D019308:Palmitic Acid; D010088:Oxidoreductases; C108138:dihydroceramide desaturase; D006867:Hydrolases; D013110:Sphingosine