Structure-function analysis of phytanoyl-CoA 2-hydroxylase mutations causing Refsum's disease

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Refsum's disease is a neurological disorder linked to phytanic acid accumulation.
• Mutations in phytanoyl-CoA 2-hydroxylase (PAHX) are a common cause of Refsum's disease.
• PAHX catalyzes the initial alpha-oxidation step in phytanic acid degradation.

Purpose of the Study:

• To characterize the enzymatic activity of wild-type and mutant PAHX.
• To investigate the role of specific amino acid residues in PAHX function.
• To evaluate the diagnostic utility of PAHX enzyme assays for Refsum's disease.

Main Methods:

• Recombinant pro and mature PAHX were produced and purified from E. coli.
• Enzyme assays were performed using phytanoyl-CoA and 2-oxoglutarate.
• Site-directed mutagenesis was used to create specific PAHX variants.
• Crystallographic data and sequence analysis guided structural predictions.

Main Results:

• PAHX requires iron(II) and 2-oxoglutarate for activity.
• Mutations H175A and D177A abolished phytanoyl-CoA hydroxylation, identifying iron-binding ligands.
• The P29S mutation led to a fully active enzyme, suggesting impaired peroxisomal targeting.
• Arg-275 mutations impaired 2-oxoglutarate binding, while other mutations caused uncoupling of reactions.

Conclusions:

• PAHX enzyme activity is crucial for phytanic acid metabolism.
• Specific mutations affect different aspects of PAHX function, including substrate binding and reaction coupling.
• PAHX enzyme assays alone are not sufficient for definitive Refsum's disease diagnosis due to complex mutation effects.