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

Douglas M Warui; Debangsu Sil; Kyung-Hoon Lee; Syam Sundar Neti; Olga A Esakova; Hayley L Knox; Carsten Krebs; Squire J Booker

doi:10.1021/acsbiomedchemau.2c00020

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

ACS Bio Med Chem Au. 2022 Oct 19;2(5):456-468. doi: 10.1021/acsbiomedchemau.2c00020. Epub 2022 Jun 13.

Authors

Douglas M Warui¹, Debangsu Sil¹, Kyung-Hoon Lee¹, Syam Sundar Neti¹, Olga A Esakova¹, Hayley L Knox¹, Carsten Krebs¹, Squire J Booker¹

Affiliation

¹ Department of Chemistry and Biochemistry and Molecular Biology and the Howard Hughes Medical Institute, The Pennsylvania State University, University Park, Pennsylvania 16802, United States.

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.