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

The last step in the biosynthesis of the lipoyl cofactor (LipCo) is the addition of two sulfur atoms at C6 and C8 of an n-octanoyl chain attached in an amide linkage to a target lysyl residue of a lipoyl carrier protein. The enzyme that catalyzes this reaction, lipoyl synthase (LipA in bacteria, and LIAS in humans), is a member of the radical S-adenosylmethionine (SAM) superfamily. As such, it requires a [4Fe-4S] cluster cofactor to cleave SAM reductively to generate two 5'-deoxyadenosyl 5'-radicals (5'-dA*) that abstract the C6 and C8 hydrogen atoms (H*) of the substrate in two distinct steps. LipAs also contain a second [4Fe-4S] cluster, termed the auxiliary cluster, degraded during turnover as the source of the attached sulfur atoms. The auxiliary cluster is ligated by three cysteines in a CX4CX5C motif and one serine residue (Ser308 in Escherichia coli) in a highly conserved R306SS308Y motif in the C-terminal region of the protein. Here, we show that Arg306 and Ser308 are absolutely required for LipCo formation. Substitution of Arg306 with Lys results in a protein that is essentially inactive due to poor substrate binding and positioning in the active site. Multiple different substitutions of Ser308 were engineered. Most notable were the S308C and S308A variants, which gave greatly diminished LipCo formation. However, the S308C variant resulted in greater production of the 6-mercaptooctanoyl peptide, an intermediate in the reaction, and the formation of a desaturated product, determined to be a 6-octenoyl group attached to the peptide substrate. Furthermore, the 3Fe cluster formed during cannibalization of the auxiliary cluster during C6 sulfur substitution in the wild-type reaction is not observed with the S308C variant. Instead, the auxiliary cluster remains tetranuclear and forms a monothiolated cross-linked species with a high-spin, S = 7/2, configuration that decays to the 6-octenoyl-containing product. Other amino acids in the RSSY motif were not essential for catalysis.