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

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

Douglas M Warui  1 , Debangsu Sil  1 , Kyung-Hoon Lee  1 , Syam Sundar Neti  1 , Olga A Esakova  1 , Hayley L Knox  1 , Carsten Krebs  1 , Squire J Booker  1
Affiliations

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

Douglas M Warui et al. ACS Bio Med Chem Au. .

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.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Proposed de novo biosynthetic scheme of lipoyl cofactor in humans.
Figure 2
Figure 2
Sequence alignment of LS proteins from E. coli (P60716), M. tuberculosis (P9WK91), T. elongatus (Q8DLC2), and humans (O43766). The conserved residues that ligate the auxiliary and the RS clusters of LS are highlighted in blue and red, respectively.
Figure 3
Figure 3
Biophysical characterization of LIAS: SDS–PAGE gel analysis of the expression and purification of LIAS on a Ni–NTA column (A), UV–vis scan of LIAS (B), Mössbauer spectrum of LIAS (C), and EPR spectrum of dithionite-reduced LIAS (D). (A) Lane 1, pre-IPTG induction; lane 2, post-IPTG induction; lane 3, crude lysate; lane 4, Ni–NTA column flow-through; lane 5, wash; lane 6; SUMO–LIAS fusion eluate; lane 7, LIAS after the SUMO tag is removed; lane 8, protein molecular weight ladder. (B) The UV–vis absorption scan of 8 μM purified LIAS showing a broad absorption at ∼410 nm, which is typical for proteins that bind [4Fe–4S] clusters. (C) The Mössbauer spectrum of 380 μM LIAS at 4.2 K, collected in the presence of a 53 mT external magnetic field applied parallel to the direction of propagation of the γ beam. The vertical bars represent the experimental spectrum, and the blue line shows the features associated with a [4Fe–4S]2+ cluster. The arrow indicates the shoulder resulting from spectral features of a site-differentiated [4Fe–4S]2+ cluster. (D) The EPR spectrum of 400 μM LIAS reduced with 4 mM dithionite and collected at 10 K with 10 mW microwave power and 0.2 mT modulation amplitude confirming bound [4Fe–4S] clusters.
Figure 4
Figure 4
Amino acid sequence alignment of E. coli NfuA (P63020) and human NFU1 (Q9UMS0) showing the two conserved cysteine residues that ligate the Fe–S cluster highlighted in red.
Figure 5
Figure 5
SDS–PAGE analysis of the expression and purification of NFU1 on a Ni–NTA column (A), UV–vis scan of 15 μM NFU1 (B), and Mössbauer spectra of NFU1 (C). (A) Lane 1, pre-IPTG induction; lane 2, post-IPTG induction; lane 3, crude lysate; lane 4, Ni–NTA column flow-through; lane 5, wash; lane 6, SUMO–NFU1 fusion eluate; lane 7, NFU1 after the SUMO tag is removed; lane 8, protein molecular weight ladder. (B) The UV–vis absorption scan spectrum of 15 μM purified NFU1 showing a broad absorption at ∼410 nm indicative of a bound [4Fe–4S] cluster. (C) The 4.2 K Mössbauer spectra of 860 μM NFU1 in the presence of a 53 mT external magnetic field applied parallel to the direction of propagation of the γ beam. The vertical bars represent the experimental spectra, and the blue line shows the features associated with a [4Fe–4S]2+ cluster.
Figure 6
Figure 6
Size-exclusion gel filtration chromatography elution profiles of holo-LIAS (100 μM, blue), holo-NFU1 (200 μM, black), and a 1:1 mixture of holo-LIAS and holo-NFU1 (100 μM each, red) (A). SDS–PAGE analysis of the chromatographed proteins: lane 1, holo-NFU1 alone; lane 2, holo-LIAS alone; lane 3, a mixture of holo-NFU1 and holo-LIAS, indicating complex formation (B). ITC binding results of LIAS titrated into NFU1, showing entropically driven binding with a dissociation constant (KD) of 0.7 ± 0.2 μM (C).
Figure 7
Figure 7
LIAS activity determinations: LIAS (10 μM) activity in the absence of NFU1 (A), in the presence of 200 μM NFU1 (B), in the presence of both 200 μM NFU1 and 5 mM sodium citrate (C), and in the presence of 200 μM NFU1 reconstituted with 34S-labeled sulfide (D). LIAS alone catalyzes about 1 turnover of lipoyl product (blue trace) with the 6-thiooctanoyl intermediate quickly reaching a steady level (black trace) (A). The inclusion of an excess of NFU1 in the LIAS reaction promotes multiple turnovers and generation of more than 5 equiv of lipoyl product (red trace), while the formation and decay of the intermediate mimics that of LIAS alone (gray trace) (B). The inclusion of 5 mM sodium citrate, a divalent metal chelator, does not significantly alter the effect of NFU1 (purple trace) compared to reactions in which citrate is omitted (red trace) (C). In the presence of NFU1 reconstituted with 34S2–, the 32S-labeled lipoyl peptide product is formed first before formation of the mixed 32S–34S (blue trace) and 34S–34S-labeled (red trace) lipoyl peptide products (D). The data in panels C and D suggest direct cluster transfer from NFU1 to LIAS during turnover. Unless otherwise noted, all activity assays included the following at their indicated final concentrations: 350 μM octanoyl peptide substrate, 0.75 mM SAM, and 10 μM SAH nucleosidase. The reactions were carried out at room temperature in a buffer that contained 50 mM HEPES, pH 7.5, and 0.25 M KCl and were initiated with a final concentration of 1 mM dithionite. The respective data shown in panels A–D are averages from assays done in triplicate, and the error bars represent one standard deviation from the mean. The 6-thiooctanoyl intermediate data were fit to an exponential equation that accounts for its formation and decay phases (A and B), while the lipoyl peptide product data were fit to a biphasic double-exponential rate of formation equation, assuming an A → B → C model, as has been previously reported for M. tuberculosis LipA (ref (72)).
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