RESUMEN
As the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues its global spread, the exploration of novel therapeutic and diagnostic strategies is still needed. The virus enters host cells by binding the angiotensin-converting enzyme 2 (ACE2) receptor through the spike protein. Here, we develop an engineered, small, stable, and catalytically inactive version of ACE2, termed miniature ACE2 (mACE2), designed to bind the spike protein with high affinity. Employing a magnetic nanoparticle-based assay, we harnessed the strong binding affinity of mACE2 to develop a sensitive and specific platform for the detection or neutralization of SARS-CoV-2. Our findings highlight the potential of engineered mACE2 as a valuable tool in the fight against SARS-CoV-2. The success of developing such a small reagent based on a piecewise molecular design serves as a proof-of-concept approach for the rapid deployment of such agents to diagnose and fight other viral diseases.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Humanos , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/química , COVID-19/virología , COVID-19/diagnóstico , Nanopartículas de Magnetita/química , Unión Proteica , Ingeniería de Proteínas , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Ferredoxins are a family of iron-sulfur (Fe-S) cluster proteins that serve as essential electron donors in numerous cellular processes that are conserved through evolution. The promiscuous nature of ferredoxins as electron donors enables them to participate in many metabolic processes including steroid, heme, vitamin D, and Fe-S cluster biosynthesis in different organisms. However, the unique natural function(s) of each of the two human ferredoxins (FDX1 and FDX2) are still poorly characterized. We recently reported that FDX1 is both a crucial regulator of copper ionophore-induced cell death and serves as an upstream regulator of cellular protein lipoylation, a mitochondrial lipid-based post-translational modification naturally occurring on four mitochondrial enzymes that are crucial for TCA cycle function. Here we show that FDX1 directly regulates protein lipoylation by binding the lipoyl synthase (LIAS) enzyme promoting its functional binding to the lipoyl carrier protein GCSH and not through indirect regulation of cellular Fe-S cluster biosynthesis. Metabolite profiling revealed that the predominant cellular metabolic outcome of FDX1 loss of function is manifested through the regulation of the four lipoylation-dependent enzymes ultimately resulting in loss of cellular respiration and sensitivity to mild glucose starvation. Transcriptional profiling established that FDX1 loss-of-function results in the induction of both compensatory metabolism-related genes and the integrated stress response, consistent with our findings that FDX1 loss-of-function is conditionally lethal. Together, our findings establish that FDX1 directly engages with LIAS, promoting its role in cellular protein lipoylation, a process essential in maintaining cell viability under low glucose conditions.
Asunto(s)
Ferredoxinas , Lipoilación , Sulfurtransferasas , Humanos , Ferredoxinas/genética , Ferredoxinas/metabolismo , Lipoilación/genética , Unión Proteica , Respiración de la Célula/genética , Proliferación Celular/genética , Metaboloma , Sulfurtransferasas/metabolismoRESUMEN
Fluoromethyl, difluoromethyl, and trifluoromethyl groups are present in numerous pharmaceuticals and agrochemicals, where they play critical roles in the efficacy and metabolic stability of these molecules. Strategies for late-stage incorporation of fluorine-containing atoms in molecules have become an important area of organic and medicinal chemistry as well as synthetic biology. Herein, we describe the synthesis and use of Te-adenosyl-L-(fluoromethyl)homotellurocysteine (FMeTeSAM), a novel and biologically relevant fluoromethylating agent. FMeTeSAM is structurally and chemically related to the universal cellular methyl donor S-adenosyl-L-methionine (SAM) and supports the robust transfer of fluoromethyl groups to oxygen, nitrogen, sulfur, and some carbon nucleophiles. FMeTeSAM is also used to fluoromethylate precursors to oxaline and daunorubicin, two complex natural products that exhibit antitumor properties.
RESUMEN
In tumors, somatic mutations of the PTEN suppressor gene are associated with advanced disease, chemotherapy resistance, and poor survival. PTEN loss of function may occur by inactivating mutation, by deletion, either affecting one copy (hemizygous loss) leading to reduced gene expression or loss of both copies (homozygous) with expression absent. Various murine models have shown that minor reductions in PTEN protein levels strongly influence tumorigenesis. Most PTEN biomarker assays dichotomize PTEN (i.e. presence vs. absence) ignoring the role of one copy loss. We performed a PTEN copy number analysis of 9793 TCGA cases from 30 different tumor types. There were 419 (4.28%) homozygous and 2484 (25.37%) hemizygous PTEN losses. Hemizygous deletions led to reduced PTEN gene expression, accompanied by increased levels of instability and aneuploidy across tumor genomes. Outcome analysis of the pan-cancer cohort showed that losing one copy of PTEN reduced survival to comparable levels as complete loss, and was associated with transcriptomic changes controlling immune response and the tumor microenvironment. Immune cell abundances were significantly altered for PTEN loss, with changes in head and neck, cervix, stomach, prostate, brain, and colon more evident in hemizygous loss tumors. These data suggest that reduced expression of PTEN in tumors with hemizygous loss leads to tumor progression and influences anticancer immune response pathways.
Asunto(s)
Evasión Inmune , Neoplasias de la Próstata , Masculino , Humanos , Animales , Ratones , Fosfohidrolasa PTEN/genética , Genoma , Neoplasias de la Próstata/patología , Próstata/patología , Genómica , Microambiente TumoralRESUMEN
Ferredoxins are a family of iron-sulfur (Fe-S) cluster proteins that serve as essential electron donors in numerous cellular processes that are conserved through evolution. The promiscuous nature of ferredoxins as electron donors enables them to participate in many metabolic processes including steroid, heme, vitamin D and Fe-S cluster biosynthesis in different organisms. However, the unique natural function(s) of each of the two human ferredoxins (FDX1 and FDX2) are still poorly characterized. We recently reported that FDX1 is both a crucial regulator of copper ionophore induced cell death and serves as an upstream regulator of cellular protein lipoylation, a mitochondrial lipid-based post translational modification naturally occurring on four mitochondrial enzymes that are crucial for TCA cycle function. Here we show that FDX1 regulates protein lipoylation by directly binding to the lipoyl synthase (LIAS) enzyme and not through indirect regulation of cellular Fe-S cluster biosynthesis. Metabolite profiling revealed that the predominant cellular metabolic outcome of FDX1 loss-of-function is manifested through the regulation of the four lipoylation-dependent enzymes ultimately resulting in loss of cellular respiration and sensitivity to mild glucose starvation. Transcriptional profiling of cells growing in either normal or low glucose conditions established that FDX1 loss-of-function results in the induction of both compensatory metabolism related genes and the integrated stress response, consistent with our findings that FDX1 loss-of-functions is conditionally lethal. Together, our findings establish that FDX1 directly engages with LIAS, promoting cellular protein lipoylation, a process essential in maintaining cell viability under low glucose conditions.
RESUMEN
The specific activity of enzymes can be altered over long timescales in cells by synonymous mutations that alter a messenger RNA molecule's sequence but not the encoded protein's primary structure. How this happens at the molecular level is unknown. Here, we use multiscale modelling of three Escherichia coli enzymes (type III chloramphenicol acetyltransferase, D-alanine-D-alanine ligase B and dihydrofolate reductase) to understand experimentally measured changes in specific activity due to synonymous mutations. The modelling involves coarse-grained simulations of protein synthesis and post-translational behaviour, all-atom simulations to test robustness and quantum mechanics/molecular mechanics calculations to characterize enzymatic function. We show that changes in codon translation rates induced by synonymous mutations cause shifts in co-translational and post-translational folding pathways that kinetically partition molecules into subpopulations that very slowly interconvert to the native, functional state. Structurally, these states resemble the native state, with localized misfolding near the active sites of the enzymes. These long-lived states exhibit reduced catalytic activity, as shown by their increased activation energies for the reactions they catalyse.
Asunto(s)
Biosíntesis de Proteínas , Mutación Silenciosa , Codón/metabolismo , ARN Mensajero/genética , Ribosomas/metabolismo , Escherichia coli/genéticaRESUMEN
Ferredoxins comprise a large family of iron-sulfur (Fe-S) proteins that shuttle electrons in diverse biological processes. Human mitochondria contain two isoforms of [2Fe-2S] ferredoxins, FDX1 (aka adrenodoxin) and FDX2, with known functions in cytochrome P450-dependent steroid transformations and Fe-S protein biogenesis. Here, we show that only FDX2, but not FDX1, is involved in Fe-S protein maturation. Vice versa, FDX1 is specific not only for steroidogenesis, but also for heme a and lipoyl cofactor biosyntheses. In the latter pathway, FDX1 provides electrons to kickstart the radical chain reaction catalyzed by lipoyl synthase. We also identified lipoylation as a target of the toxic antitumor copper ionophore elesclomol. Finally, the striking target specificity of each ferredoxin was assigned to small conserved sequence motifs. Swapping these motifs changed the target specificity of these electron donors. Together, our findings identify new biochemical tasks of mitochondrial ferredoxins and provide structural insights into their functional specificity.
Asunto(s)
Ferredoxinas , Proteínas Hierro-Azufre , Humanos , Isoformas de Proteínas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Mitocondrias/metabolismo , Proteínas Hierro-Azufre/metabolismoRESUMEN
Lipoic acid is an eight-carbon sulfur-containing biomolecule that functions primarily as a cofactor in several multienzyme complexes. It is biosynthesized as an attachment to a specific lysyl residue on one of the subunits of these multienzyme complexes. In Escherichia coli and many other organisms, this biosynthetic pathway involves two dedicated proteins: octanoyltransferase (LipB) and lipoyl synthase (LipA). LipB transfers an n-octanoyl chain from the octanoyl-acyl carrier protein to the target lysyl residue, and then, LipA attaches two sulfur atoms (one at C6 and one at C8) to give the final lipoyl cofactor. All classical lipoyl synthases (LSs) are radical S-adenosylmethionine (SAM) enzymes, which use an [Fe4S4] cluster to reductively cleave SAM to generate a 5'-deoxyadenosyl 5'-radical. Classical LSs also contain a second [Fe4S4] cluster that serves as the source of both appended sulfur atoms. Recently, a novel pathway for generating the lipoyl cofactor was reported. This pathway replaces the canonical LS with two proteins, LipS1 and LipS2, which act together to catalyze formation of the lipoyl cofactor. In this work, we further characterize LipS1 and LipS2 biochemically and spectroscopically. Although LipS1 and LipS2 were previously annotated as biotin synthases, we show that both proteins, unlike E. coli biotin synthase, contain two [Fe4S4] clusters. We identify the cluster ligands to both iron-sulfur clusters in both proteins and show that LipS2 acts only on an octanoyl-containing substrate, while LipS1 acts only on an 8-mercaptooctanoyl-containing substrate. Therefore, similarly to E. coli biotin synthase and in contrast to E. coli LipA, sulfur attachment takes place initially at the terminal carbon (C8) and then at the C6 methylene carbon.
RESUMEN
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.
RESUMEN
X-linked agammaglobulinemia (XLA) is an inborn error of immunity characterized by insufficient production of immunoglobulins and lack of measurable antibody response to vaccines. The rise of novel infections limits the protective effect of immunoglobulin replacement in immunodeficient patients though. While XLA patients are not expected to mount an antibody response to COVID-19 vaccination, it has been demonstrated that XLA patients can mount a T-cell response to COVID-19 vaccines, similar to the influenza vaccine. We present three patients with XLA who received an mRNA COVID-19 vaccine. One patient demonstrated positive antibody response. Many XLA patients do not receive routine vaccinations due to ongoing immunoglobulin replacement therapy and lack of native antibody production, but in addition to T-cell response to vaccination, select XLA patients may mount a positive antibody response. Therefore, COVID-19 vaccination should be encouraged for all XLA patients.
Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , Agammaglobulinemia , COVID-19/prevención & control , Enfermedades Genéticas Ligadas al Cromosoma X , Humanos , Inmunoglobulinas , ARN Mensajero , VacunaciónRESUMEN
Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.
Asunto(s)
Proteínas Arqueales , Éteres de Glicerilo , Lípidos de la Membrana , Methanocaldococcus , Proteínas Arqueales/química , Proteínas Arqueales/aislamiento & purificación , Proteínas Arqueales/metabolismo , Carbono/química , Carbono/metabolismo , Glicerol/química , Glicerol/metabolismo , Éteres de Glicerilo/química , Éteres de Glicerilo/metabolismo , Lípidos de la Membrana/biosíntesis , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Methanocaldococcus/química , Methanocaldococcus/enzimología , Methanocaldococcus/metabolismo , S-Adenosilmetionina/metabolismo , Terpenos/química , Terpenos/metabolismoRESUMEN
Cobalamin-dependent radical S-adenosylmethionine (SAM) methylases catalyze key steps in the biosynthesis of numerous biomolecules, including protein cofactors, antibiotics, herbicides, and other natural products, but have remained a relatively understudied subclass of radical SAM enzymes due to their inherent insolubility upon overproduction in Escherichia coli. These enzymes contain two cofactors: a [4Fe-4S] cluster that is ligated by three cysteine residues, and a cobalamin cofactor typically bound by residues in the N-terminal portion of the enzyme. Recent advances in the expression and purification of these enzymes in their active states and with both cofactors present has allowed for more detailed biochemical studies as well as structure determination by X-ray crystallography. Herein, we use KsTsrM and TokK to highlight methods for the structural characterization of cobalamin-dependent radical SAM (RS) enzymes and describe recent advances in in the overproduction and purification of these enzymes.
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S-Adenosilmetionina , Vitamina B 12 , Cristalografía por Rayos X , Escherichia coli/metabolismo , Metiltransferasas/metabolismo , S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismoRESUMEN
Nosiheptide is a ribosomally produced and post-translationally modified thiopeptide antibiotic that displays potent antibacterial activity in vitro, especially against Gram-positive pathogens. It comprises a core peptide macrocycle that contains multiple thiazole rings, dehydrated serine and threonine residues, a tri-substituted 3-hydroxypyridine ring and several other modifications. Among these additional modifications includes a 3,4-dimethyl-2-indolic acid (DMIA) moiety that bridges Glu6 and Cys8 of the core peptide to form a second smaller ring system. This side-ring system is formed by the action of NosN, a radical S-adenosylmethionine (SAM) enzyme that falls within the class C radical SAM methylase (RSMT) family. However, the true function of NosN is to transfer a methylene group from the methyl moiety of SAM to C4 of 3-methylindolic acid (MIA) attached in a thioester linkage to Cys8 of the core peptide to set up a highly electrophilic species. This species is then trapped by the side chain of Glu6, resulting in formation of a lactone and the side-ring system. The NosN reaction requires two simultaneously bound molecules of SAM. The first, SAMI, is cleaved to generate a 5'-deoxyadenosyl 5'-radical, which abstracts a hydrogen atom from the methyl group of the second molecule of SAM, SAMII. The resulting SAMII radical is believed to add to C4 of MIA, affording a radical intermediate on the MIA substrate. Herein we describe synthetic approaches that allow detection of this radical by electron paramagnetic resonance (EPR) spectroscopy.
Asunto(s)
Proteínas Hierro-Azufre , S-Adenosilmetionina , Antibacterianos , Catálisis , Proteínas Hierro-Azufre/química , Metiltransferasas/metabolismo , Péptidos/química , S-Adenosilmetionina/metabolismoRESUMEN
Maturation of [FeFe]-hydrogenase (HydA) involves synthesis of a CO, CN- , and dithiomethylamine (DTMA)-coordinated 2Fe subcluster that is inserted into HydA to make the active hydrogenase. This process requires three maturation enzymes: the radical S-adenosyl-l-methionine (SAM) enzymes HydE and HydG, and the GTPase HydF. In vitro maturation with purified maturation enzymes has been possible only when clarified cell lysate was added, with the lysate presumably providing essential components for DTMA synthesis and delivery. Here we report maturation of [FeFe]-hydrogenase using a fully defined system that includes components of the glycine cleavage system (GCS), but no cell lysate. Our results reveal for the first time an essential role for the aminomethyl-lipoyl-H-protein of the GCS in hydrogenase maturation and the synthesis of the DTMA ligand of the H-cluster. In addition, we show that ammonia is the source of the bridgehead nitrogen of DTMA.
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Hidrogenasas , Proteínas Hierro-Azufre , Espectroscopía de Resonancia por Spin del Electrón , Hidrogenasas/metabolismo , Ligandos , S-AdenosilmetioninaRESUMEN
Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.
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Carbapenémicos/biosíntesis , Metiltransferasas/química , Metiltransferasas/metabolismo , S-Adenosilmetionina/metabolismo , Streptomyces/enzimología , Tienamicinas/biosíntesis , Vitamina B 12/metabolismo , Sitios de Unión , Biocatálisis , Coenzimas/metabolismo , Cristalografía por Rayos X , Cinética , Metilación , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Streptomyces/metabolismo , Inhibidores de beta-Lactamasas/metabolismo , beta-Lactamasas/química , beta-Lactamasas/metabolismoRESUMEN
Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon-anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity1-4. The ms2i6A modification is installed onto isopentenyladenosine (i6A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe4S4]RS cluster used in the reductive cleavage of SAM to form a 5'-deoxyadenosyl 5'-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6-9 in sulfur transfer to C2 of i6A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5'-deoxyadenosyl 5'-radical, which abstracts the C2 hydrogen of the substrate but only after C2 has undergone rehybridization from sp2 to sp3. This work advances our understanding of how enzymes functionalize inert C-H bonds with sulfur.
Asunto(s)
Bacteroides/enzimología , Metiltransferasas/química , ARN de Transferencia/química , ARN de Transferencia/metabolismo , S-Adenosilmetionina/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Sulfurtransferasas/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Sitios de Unión , Biocatálisis , Isopenteniladenosina/metabolismo , Metiltransferasas/metabolismo , Modelos Moleculares , Dominios Proteicos , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Especificidad por Sustrato , Sulfurtransferasas/metabolismoRESUMEN
Lipoic acid is an essential sulfur-containing cofactor used by several multienzyme complexes involved in energy metabolism and the breakdown of certain amino acids. It is composed of n-octanoic acid with sulfur atoms appended at C6 and C8. Lipoic acid is biosynthesized de novo in its cofactor form, in which it is covalently bound in an amide linkage to a target lysyl residue on a lipoyl carrier protein (LCP). The n-octanoyl moiety of the cofactor is derived from type 2 fatty acid biosynthesis and is transferred to an LCP to afford an octanoyllysyl amino acid. Next, lipoyl synthase (LipA in bacteria) catalyzes the attachment of the two sulfur atoms to afford the intact cofactor. LipA is a radical S-adenosylmethionine (SAM) enzyme that contains two [4Fe-4S] clusters. One [4Fe-4S] cluster is used to facilitate a reductive cleavage of SAM to render the highly oxidizing 5'-deoxyadenosyl 5'-radical needed to abstract C6 and C8 hydrogen atoms to allow for sulfur attachment. By contrast, the second cluster is the sulfur source, necessitating its destruction during turnover. In Escherichia coli, this auxiliary cluster can be restored after each turnover by NfuA or IscU, which are two iron-sulfur cluster carrier proteins that are implicated in iron-sulfur cluster biogenesis. In this chapter, we describe methods for purifying and characterizing LipA and NfuA from Mycobacterium tuberculosis, a human pathogen for which endogenously synthesized lipoic acid is essential. These studies provide the foundation for assessing lipoic acid biosynthesis as a potential target for the design of novel antituberculosis agents.