Purification, molecular cloning, and expression of 2-hydroxyphytanoyl-CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzesthe carbon-carbon bond cleavage during alpha-oxidation of 3-methyl-branched fatty acids

In the third step of the Lu-oxidation of 3-methyl-branched fatty acids such as phytanic acid, a 2-hydroxy-3-methylacyl-CoA is cleaved into formyl-CoA and a 2-methyl-branched fatty aldehyde. The cleavage enzyme was purified fr om the matrix protein fraction of rat liver peroxisomes and identified as a protein made up of four identical subunits of 63 kDa, Its activity proved to depend on Mg2+ and thiamine pyrophosphate, a hitherto unrecognized cofac tor of a-oxidation. Formyl-CoA and 2-methylpentadecanal were identified as reaction products when the purified enzyme was incubated with 2-hydroxy-3-m ethylhexadecanoyl-CoA as the substrate. Hence the enzyme catalyzes a carbon -carbon cleavage, and we propose calling it 2-hydroxyphytanoyl-CoA lyase. S equences derived from tryptic peptides of the purified rat protein were use d as queries to recover human expressed sequence tags from the databases. T he composite cDNA sequence of the human lyase contained an ORF of 1,734 bas es that encodes a polypeptide with a calculated molecular mass of 63,732 Da . Recombinant human protein, expressed in mammalian cells, exhibited lyase activity. The lyase displayed homology to a putative Caenorhabditis elegans protein that resembles bacterial oxalyl-CoA decarboxylases. Similarly to t he decarboxylases, a thiamine pyrophosphate-binding consensus domain was pr esent in the C-terminal part of the lyase, Although no peroxisome targeting signal, neither 1 nor 2, was apparent, transfection experiments with const ructs encoding green fluorescent protein fused to the full-length lyase or its C-terminal pentapeptide indicated that the C terminus of the lyase repr esents a peroxisome targeting signal 1 variant.