Role of thiamine pyrophosphate in oligomerisation, functioning and import of peroxisomal 2-hydroxyacyl-CoA lyase

During peroxisomal α-oxidation, the CoA-esters of phytanic acid and 2-hydroxylated straight chain fatty acids are cleaved into a (n-1) fatty aldehyde and formyl-CoA by 2-hydroxyacyl-CoA lyase (HACL1). HACL1 is imported into peroxisomes via the PEX5/PTS1 pathway, and so far, it is the only known peroxisomal TPP-dependent enzyme in mammals. In this study, the effect of mutations in the TPP-binding domain of HACL1 on enzyme activity, subcellular localisation and oligomerisation was investigated. Mutations of the aspartate 455 and serine 456 residues within the TPP binding domain of the human HACL1 did not affect the targeting upon expression in transfected CHO cells, although enzyme activity was abolished. Gel filtration of native and mutated N-His6-fusions, expressed in yeast, revealed that the mutations did not influence the oligomerisation of the (apo)enzyme. Subcellular fractionation of yeast cells expressing HACL1 showed that the lyase activity sedimented at high density in a Nycodenz gradient. In these fractions TPP could be measured, but not when mutated HACL1 was expressed, although the recombinant enzyme was still targeted to peroxisomes. These findings indicate that the binding of TPP is not required for peroxisomal targeting and correct folding of HACL1, in contrast to other TPP-dependent enzymes, and suggest that transport of TPP into peroxisomes is dependent on HACL1 import, without requirement of a specific solute transporter.

► In this study, the cofactor binding site of human 2-hydroxyacyl-CoA lyase (HACL1), an enzyme involved in alpha-oxidation was investigated.
► Mutational analysis revealed the importance of aspartate 455 and serine 456 for binding TPP and for enzymatic activity.
► Oligomerisation of HACL1 does not require TPP binding.
► Targeting of HACL1 to peroxisomes is not dependent on cofactor binding.
► Transport of TPP into peroxisomes is likely dependent on HACL1 import.