Phytanoyl-CoA hydroxylase: recognition of 3-methyl-branched acyl-coAs and requirement for GTP or ATP and Mg(2+) in addition to its known hydroxylation cofactors.
Phytanoyl-CoA hydroxylase is a peroxisomal alpha-oxidation enzyme that catalyzes the 2-hydroxylation of 3-methyl-branched acyl-CoAs. A polyhistidine-tagged human phytanoyl-CoA hydroxylase was expressed in E. coli and subsequently purified as an active protein. The recombinant enzyme required GTP or ATP and Mg(2+), in addition to its known cofactors Fe(2+), 2-oxoglutarate, and ascorbate. The enzyme was active towards phytanoyl-CoA and 3-methylhexadecanoyl-CoA, but not towards 3-methylhexadecanoic acid. Racemic, R- and S-3-methylhexadecanoyl-CoA were equally well hydroxylated. Hydroxylation of R- and S-3-methylhexadecanoyl-CoA yielded the (2S, 3R) and (2R,3S) isomers of 2-hydroxy-3-methylhexadecanoyl-CoA, respectively. Human phytanoyl-CoA hydroxylase did not show any activity towards 2-methyl- and 4-methyl-branched acyl-CoAs or towards long and very long straight chain acyl-CoAs, excluding a possible role for the enzyme in the formation of 2-hydroxylated and odd-numbered straight chain fatty acids, which are abundantly present in brain. In conclusion, we report the unexpected requirement for ATP or GTP and Mg(2+) of phytanoyl-CoA hydroxylase in addition to the known hydroxylation cofactors. Due to the fact that straight chain fatty acyl-CoAs are not a substrate for phytanoyl-CoA hydroxylase, 2-hydroxylation of fatty acids in brain can be allocated to a different enzyme/pathway.
Croes
K
K
Department Moleculaire Celbiologie, Afdeling Farmacologie, Katholieke Universiteit Leuven, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium.
Foulon
V
V
Casteels
M
M
Van Veldhoven
P P
PP
Mannaerts
G P
GP
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007656:Ketoglutaric Acids / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D010169:Palmitic Acids / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D002413:Cations, Divalent; D005296:Ferrous Compounds; D007656:Ketoglutaric Acids; D010169:Palmitic Acids; D011994:Recombinant Proteins; C034840:3-methylhexadecanoic acid; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006899:Mixed Function Oxygenases; C102623:PHYH protein, human; D008274:Magnesium