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

Reference ID

54964

Title

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

Source

Proc Natl Acad Sci U S A, 1999, vol. 96, pp. 10039-10044

Authors (7)

Foulon-V	Antonenkov-V-D
Croes-K	Waelkens-E
Mannaerts-G-P	Van-Veldhoven-P-P
Casteels-M

Abstract

In the third step of the alpha-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 from 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
Mg(2+) and thiamine pyrophosphate, a hitherto unrecognized cofactor of alpha-
oxidation. Formyl-CoA and 2-methylpentadecanal were identified as reaction
products when the purified enzyme was incubated with 2-hydroxy-3-methylhexadecanoyl-
CoA as the substrate. Hence the enzyme catalyzes a carbon-carbon cleavage, and
we propose calling it 2-hydroxyphytanoyl-CoA lyase. Sequences derived from
tryptic peptides of the purified rat protein were used as queries to recover
human expressed sequence tags from the databases. The composite cDNA sequence of
the human lyase contained an ORF of 1,734 bases 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 the decarboxylases, a thiamine pyrophosphate-
binding consensus domain was present in the C-terminal part of the lyase.
Although no peroxisome targeting signal, neither 1 nor 2, was apparent,
transfection experiments with constructs encoding green fluorescent protein
fused to the full-length lyase or its C-terminal pentapeptide indicated that the
C terminus of the lyase represents a peroxisome targeting signal 1 variant.