Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

Abstract

Summary Statement: The biosynthesis of lipoyl cofactors requires two lipoyl synthase mediated sulfur insertions. We report the crystal structures of a lipoyl synthase complexed with S-adenosylhomocysteine or 5'-methylthioadenosine. Models based on these structures identify likely substrate binding sites.Keywords: radical SAM, cofactors, crystal structure, enzyme catalysis, sulfur Abbreviations used: LipA, lipoyl synthase; BioB, biotin synthase; SAM, Sadenosylmethionine; LCD, lipoyl carrier domain; MTA, 5'-methylthioadenosine; RS, radical SAM; ACP, acyl carrier protein; SsLipA, Sulfolobus solfataricus LipA; TeLipA2 Thermosynechococcus elongatus LipA2; Ec, Escherichia coli; 5'-dA, 5'-deoxyadenosine. 2 ABSTRACTLipoyl cofactors are essential for living organisms and are produced by the insertion of two sulfur atoms into the relatively unreactive C-H bonds of an octanoyl substrate. This reaction requires lipoyl synthase, a member of the radical SAM enzyme superfamily. Herein we present crystal structures of lipoyl synthase with two [4Fe-4S] clusters bound at opposite ends of the TIM barrel, the usual fold of the radical SAM superfamily. The cluster required for reductive SAM cleavage conserves the features of the radical SAM superfamily, but the auxiliary cluster is bound by a CX 4 CX 5 C motif unique to lipoyl synthase. The fourth ligand to the auxiliary cluster is an extremely unusual serine residue. Site directed mutants show this conserved serine ligand is essential for the sulfur insertion steps. One crystallized LipA complex contains MTA, a breakdown product of SAM, bound in the likely SAM binding site. Modelling has identified an 18 Å deep channel, well-proportioned to accommodate an octanoyl substrate. These results suggest the auxiliary cluster is the likely sulfur donor, but access to a sulfide ion for the second sulfur insertion reaction requires the loss of an iron atom from the auxiliary cluster, which the serine ligand may enabled.3

Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

“…Moreover, LipA was sometimes purified under aerobic conditions. The demonstration that LipA contains two distinct [4Fe-4S] clusters per polypeptide (105) has been confirmed by a recent crystal structure (106).…”

“…Very recently, the first crystal structure of a lipoyl synthase, that of the thermophilic cyanobacterium Thermosynechococcus elongates, was reported by Harmer and coworkers (106). The structure shows the expected two [4Fe-4S] clusters per monomer (Fig.…”

“…The authors report that the replacement of this serine residue with cysteine or alanine results in complete loss of the ability to synthesize lipoyl peptide. Moreover, the mutant proteins showed significantly less uncoupled SAM turnover than the wild-type protein (106). Since uncoupled SAM turnover is the function of the radical SAM [4Fe-4S] cluster, it seems that the two clusters must somehow communicate.…”

“…One of two LipA [4Fe-4S] clusters is coordinated by the radical SAM CXXXCXXC motif that functions in 5=-dA radical generation. The second cluster is coordinated by a CXXXXCX XXXXC motif plus a serine residue that is found only in lipoyl synthases (106). This second cluster (now generally called the auxiliary cluster) was suggested to be the source of the sulfur atoms (as sulfide).…”

“…The Thermosynechococcus elongates LipA structure (106). The auxiliary cluster (Aux) is to the left, and the radical SAM (RS) cluster is to the right.…”