Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide

Area of Science:

• Biochemistry
• Enzymology
• Metalloenzymology

Background:

• Lipoyl synthase (LS) is a radical SAM enzyme essential for lipoic acid cofactor biosynthesis.
• The enzyme uses S-adenosyl-l-methionine (SAM) to generate a 5'-deoxyadenosyl radical (5'-dA(*)) for hydrogen abstraction.
• The identity of the sulfur donor in the lipoylation reaction remains elusive.

Purpose of the Study:

• To investigate the role and stoichiometry of iron-sulfur ([4Fe-4S]) clusters in Escherichia coli lipoyl synthase.
• To elucidate the structural basis for the two distinct [4Fe-4S] clusters within lipoyl synthase.
• To determine the contribution of these clusters to enzyme activity and cofactor formation.

Main Methods:

• Expression of wild-type and mutant lipoyl synthase in E. coli with an iron-sulfur cluster biosynthesis operon.
• Biochemical characterization including iron and sulfide quantification.
• Spectroscopic analysis using Mössbauer and EPR spectroscopy.
• Enzymatic assays measuring 5'-deoxyadenosine (5'-dA) and lipoylated H-protein formation.

Main Results:

• Wild-type lipoyl synthase contains two distinct [4Fe-4S] clusters, each ligated by specific cysteine motifs.
• Mutational analysis of conserved cysteine residues abolished enzyme activity, confirming the necessity of both clusters.
• Reconstitution studies and spectroscopy indicate the presence of approximately two [4Fe-4S](2+) clusters per active enzyme molecule.

Conclusions:

• Lipoyl synthase employs two unique [4Fe-4S] clusters, essential for its catalytic mechanism involving radical generation and sulfur insertion.
• The distinct cysteine ligation motifs dictate the specific roles and properties of each iron-sulfur cluster.
• This study provides critical insights into the structural and mechanistic aspects of lipoic acid biosynthesis via radical SAM enzymes.