Amino Acids in the RSSY Motif of Lipoyl Synthase Control Substrate Binding and Reactivity^†

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Lipoyl cofactor (LipCo) biosynthesis involves the addition of sulfur atoms to an octanoyl chain on a carrier protein.
• Lipoyl synthase (LipA/LIAS), a radical S-adenosylmethionine (SAM) enzyme, catalyzes this final step using iron-sulfur clusters.
• LipA utilizes a [4Fe-4S] cluster for SAM cleavage and another auxiliary [4Fe-4S] cluster as the sulfur source.

Purpose of the Study:

• To investigate the essentiality of the conserved R ³⁰⁶ SS ³⁰⁸ Y motif in lipoyl synthase function.
• To elucidate the roles of Arg306 and Ser308 in substrate binding, positioning, and catalysis.
• To characterize the impact of Ser308 mutations on auxiliary cluster behavior and reaction intermediates.

Main Methods:

• Site-directed mutagenesis was employed to generate variants of Arg306 and Ser308 in lipoyl synthase.
• Enzyme activity assays were performed to measure lipoyl cofactor formation.
• Spectroscopic methods and product analysis were used to characterize reaction intermediates and products.

Main Results:

• Arg306 and Ser308 are critical for lipoyl cofactor formation; Arg306 substitutions abolish activity.
• Ser308 substitutions, particularly S308C, lead to reduced LipCo formation but increased intermediate production (6-mercaptooctanoyl peptide).
• The S308C variant forms a unique monothiolated cross-linked auxiliary cluster species and a desaturated 6-octenoyl product, unlike wild-type.

Conclusions:

• The R ³⁰⁶ SS ³⁰⁸ Y motif, specifically Arg306 and Ser308, is indispensable for lipoyl synthase catalysis.
• Ser308 plays a crucial role in modulating auxiliary cluster degradation and product formation pathways.
• Mutational analysis reveals distinct mechanistic roles for key residues in the lipoyl synthase reaction.