Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• 5-Aminolevulinate synthase (ALAS) is a key enzyme in heme biosynthesis, crucial for non-plant eukaryotes and some prokaryotes.
• ALAS functions as a homodimer, requiring pyridoxal 5'-phosphate (PLP) as a cofactor.
• While active site residues are studied, the role of polypeptide chain arrangement in ALAS folding and function remains less understood.

Purpose of the Study:

• To investigate the importance of polypeptide chain continuity for 5-aminolevulinate synthase folding, structure, and function.
• To explore the impact of circular permutation on ALAS activity and assembly.
• To determine if alternative folding pathways exist for ALAS.

Main Methods:

• Construction of circularly permuted 5-aminolevulinate synthase variants via rational design and random library screening.
• Analysis of active variants for sequence differences, circular permutations, and altered termini.
• Comparison of wild-type and variant structures, including secondary structure arrangement and cofactor-binding site.

Main Results:

• Identified 21 active ALAS variants, 9 with unique circular permutations.
• New termini in variants disrupted secondary structures but did not prevent folding, PLP binding, or subunit assembly.
• The order of catalytic and glycine-binding domains was found to be irrelevant for enzyme function.
• Circularly permuted variants showed altered secondary structure arrangements and cofactor-binding environments despite similar predicted tertiary structures.

Conclusions:

• Polypeptide chain continuity is not a strict requirement for 5-aminolevulinate synthase proper folding, cofactor binding, or homodimerization.
• The sequential arrangement of secondary structure elements and functional domains can be altered without compromising enzyme activity.
• These findings suggest that 5-aminolevulinate synthase can achieve its functional structure through multiple or alternative folding pathways.