In Vitro Demonstration of Human Lipoyl Synthase Catalytic Activity in the Presence of NFU1

Abstract

Lipoyl synthase (LS) catalyzes the last step in the biosynthesis of the lipoyl cofactor, which is the attachment of sulfur atoms at C6 and C8 of an n-octanoyllysyl side chain of a lipoyl carrier protein (LCP). The protein is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes, which use SAM as a precursor to a 5′-deoxyadenosyl 5′-radical (5′-dA·). The role of the 5′-dA· in the LS reaction is to abstract hydrogen atoms from C6 and C8 of the octanoyl moiety of the substrate to initiate subsequent sulfur attachment. All radical SAM enzymes have at least one [4Fe–4S] cluster that is used in the reductive cleavage of SAM to generate the 5′-dA·; however, LSs contain an additional auxiliary [4Fe–4S] cluster from which sulfur atoms are extracted during turnover, leading to degradation of the cluster. Therefore, these enzymes catalyze only 1 turnover in the absence of a system that restores the auxiliary cluster. In Escherichia coli, the auxiliary cluster of LS can be regenerated by the iron–sulfur (Fe–S) cluster carrier protein NfuA as fast as catalysis takes place, and less efficiently by IscU. NFU1 is the human ortholog of E. coli NfuA and has been shown to interact directly with human LS (i.e., LIAS) in yeast two-hybrid analyses. Herein, we show that NFU1 and LIAS form a tight complex in vitro and that NFU1 can efficiently restore the auxiliary cluster of LIAS during turnover. We also show that BOLA3, previously identified as being critical in the biosynthesis of the lipoyl cofactor in humans and Saccharomyces cerevisiae, has no direct effect on Fe–S cluster transfer from NFU1 or GLRX5 to LIAS. Further, we show that ISCA1 and ISCA2 can enhance LIAS turnover, but only slightly.

FDX1 regulates cellular protein lipoylation through direct binding to LIAS

“…This is not due to changes in total protein levels of the lipoylated proteins (as shown for DLAT). Cellular protein lipoylation is regulated by the Fe-S cluster enzyme LIAS (25,26) that uses the sulfurs from its auxiliary Fe-S cluster to facilitate the double sulfur insertion to the sixth and eighth carbons of the octanoyl precursor of lipoic acid (18). This dependency of LIAS on Fe-S clusters for function has been shown in genetic models where perturbations to Fe-S cluster biosynthesis results in reduced levels of LIAS and consequently reduced levels of protein lipoylation (27,28).…”

“…During purification, the SUMO tag was cleaved from the fusion protein affording pure LIAS with a Gly-His appendage at the N-terminus. Both proteins were overexpressed, purified and their respective iron-sulfur clusters reconstituted following procedures as previously published for LIAS with no further modifications (18).…”

“…Second, two sulfurs are added at C-6 and C-8 positions by the lipoyl synthase (LIAS) enzyme to form the lipoylated form of GCSH. In this reaction the sulfur atoms are provided by the auxiliary Fe-S cluster of LIAS, which requires Fe-S cluster reconstitution on LIAS after each catalytic cycle of lipoic acid synthesis (18)(19)(20). Therefore, any perturbation in cellular Fe-S cluster biosynthesis will result in reduced ability of the cell to replenish the auxiliary Fe-S cluster of LIAS after each cycle of lipoic acid synthesis, and ultimately reduced levels of protein lipoylation.…”

Twenty Years of Radical SAM! The Genesis of the Superfamily

“…An Article in this virtual issue from the Booker laboratory describes the characterization of the human form of LipA, called LIAS, as well as the human NfuA analogue, NFU1. This Article reports that NFU1 binds tightly to LIAS and, like NfuA’s effect on LipA, can regenerate the auxiliary cluster of LIAS, allowing the protein to conduct multiple turnovers . A second Article contributed by the Booker laboratory describes studies on a completely new system for generating the lipoyl cofactor that is found predominantly in archaea.…”

“…This Article reports that NFU1 binds tightly to LIAS and, like NfuA’s effect on LipA, can regenerate the auxiliary cluster of LIAS, allowing the protein to conduct multiple turnovers. 100 A second Article contributed by the Booker laboratory describes studies on a completely new system for generating the lipoyl cofactor that is found predominantly in archaea. Unlike the canonical system (i.e., LipA and LIAS), which requires only a protein composed of a single polypeptide, this new system requires two proteins to insert both sulfurs.…”