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

Abstract

In the third step of the ␣-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 ␣-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.

RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1

“…A gender dependency, so far not reported, was found (106 ± 12 mU/g liver in females, n = 4, and 71 ± 19 mU/g liver in males, n = 6). The values obtained were comparable to those previously measured in rodent liver homogenates using long-chain 2-hydroxyacylCoA as substrate ( 5,6,23 ). Linearity of HACL1 activity in mouse liver homogenates is limited to 5 min (at 1 mg (40 M).…”

A Human CTL Recognizes a Caspase-8-Derived Peptide on Autologous HLA-B*3503 Molecules and Two Unrelated Peptides on Allogeneic HLA-B*3501 Molecules

“…cDNAs 167 and 576 correspond to 2-hydroxyphytanoyl-CoA lyase (accession no. HSA131753), which is also expressed in normal tissues and catalyzes the carbon-carbon bond cleavage during oxidation of some fatty acids (28). The peptide recognized by CTL 121 was identified with the same strategy as described for squalene synthase.…”

Biochemistry and genetics of inherited disorders of peroxisomal fatty acid metabolism

“…In addition to long-chain FAs, various other carboxylates can be -hydroxylated, and depending on the chain length, substituents, and double bonds, different CYP4, belonging to the F and A subfamilies, are involved ( 165,166 ). The -hydroxy-FAs are further oxidized to dicarboxylic acids, which are excreted with or without prior shortening via hepatic lipids (C [24][25][26][27][28][29][30] with fi ve and six double bonds), although DHA levels were normal ( 160 ). Similarly, in mice lacking MPF2, lipid accumulation is observed in testis, cerebellum, and retina, and abnormal very long PUFA were seen in the FA profi les of testis ( 161 ).…”