Metabolic Functions of Biliverdin IXß Reductase in Redox-Regulated Hematopoietic Cell Fate.

Cytoprotective heme oxygenases derivatize heme to generate carbon monoxide, ferrous iron, and isomeric biliverdins, followed by rapid NAD(P)H-dependent biliverdin reduction to the antioxidant bilirubin. Recent studies have implicated biliverdin IXß reductase (BLVRB) in a redox-regulated mechanism of hematopoietic lineage fate restricted to megakaryocyte and erythroid development, a function distinct and non-overlapping from the BLVRA (biliverdin IXα reductase) homologue. In this review, we focus on recent progress in BLVRB biochemistry and genetics, highlighting human, murine, and cell-based studies that position BLVRB-regulated redox function (or ROS accumulation) as a developmentally tuned trigger that governs megakaryocyte/erythroid lineage fate arising from hematopoietic stem cells. BLVRB crystallographic and thermodynamic studies have elucidated critical determinants of substrate utilization, redox coupling and cytoprotection, and have established that inhibitors and substrates bind within the single-Rossmann fold. These advances provide unique opportunities for the development of BLVRB-selective redox inhibitors as novel cellular targets that retain potential for therapeutic applicability in hematopoietic (and other) disorders.

H-NOX Regulates Biofilm Formation in Agrobacterium Vitis in Response to NO.

Transitions between motile and biofilm lifestyles are highly regulated and fundamental to microbial pathogenesis. H-NOX (heme-nitric oxide/oxygen-binding domain) is a key regulator of bacterial communal behaviors, such as biofilm formation. A predicted bifunctional cyclic di-GMP metabolizing enzyme, composed of diguanylate cyclase and phosphodiesterase (PDE) domains (avi_3097), is annotated downstream of an hnoX gene in Agrobacterium vitis S4. Here, we demonstrate that avH-NOX is a nitric oxide (NO)-binding hemoprotein that binds to and regulates the activity of avi_3097 (avHaCE; H-NOX-associated cyclic di-GMP processing enzyme). Kinetic analysis of avHaCE indicates a ∼four-fold increase in PDE activity in the presence of NO-bound avH-NOX. Biofilm analysis with crystal violet staining reveals that low concentrations of NO reduce biofilm growth in the wild-type A. vitis S4 strain, but the mutant ΔhnoX strain has no NO phenotype, suggesting that H-NOX is responsible for the NO biofilm phenotype in A. vitis. Together, these data indicate that avH-NOX enhances cyclic di-GMP degradation to reduce biofilm formation in response to NO in A. vitis.

Genetic pathways regulating hematopoietic lineage speciation: Factorial latent variable model analysis of single cell transcriptome.

Genetic pathways regulating hematopoietic lineage commitment at critical stages of development remain incompletely characterized.  To better delineate genetic sources of variability regulating cellular speciation during steady-state hematopoiesis, we applied a factorial single-cell latent variable model (f-scLVM) to decompose single-cell transcriptome heterogeneity into interpretable biological factors (refined pathway annotations or gene sets without annotation) dynamically regulating cell fate.  Hematopoietic single cell transcriptomic raw sequencing data extracted from 1,920 hematopoietic stem and progenitor cells (HSPCs) derived from 12-week-old female mice were used for data analysis and model development. These single cell RNA sequencing data were subsequently analyzed using the factorial single-cell latent variable model (f-scLVM), with their heterogeneity decomposed into interpretable biological factors. The top biological factors underlying the basal hematopoiesis were subsequently identified for the aggregate, and lineage-restricted (myeloid, megakaryocyte, erythroid) progenitor cells. For a subset of factors, data were independently verified experimentally in a companion research paper [1]. These data facilitate the identification of novel subpopulations and adjust gene sets to discover new marker genes and hidden confounding factors driving basal hematopoiesis.

Divergent erythroid megakaryocyte fates in Blvrb-deficient mice establish non-overlapping cytoprotective functions during stress hematopoiesis.

Cytoprotective mechanisms of heme oxygenases function by derivatizing heme to generate carbon monoxide, ferrous iron, and isomeric biliverdins, followed by rapid NAD(P)H-dependent biliverdin reduction to the antioxidant bilirubin using two non-overlapping biliverdin reductases that display biliverdin isomer-restricted redox activity. Although cytoprotective functions of heme oxygenases are widely recognized, concomitant effects of downstream biliverdin reductases remain incomplete. A computational model predicated on murine hematopoietic single-cell transcriptomic data identified Blvrb as a biological driver linked to the tumor necrosis factor stress pathway as a predominant source of variation defining hematopoietic cell heterogeneity. In vivo studies using Blvrb-deficient mice established the dispensable role of Blvrb in steady-state hematopoiesis, although model validation using aged Blvrb-deficient mice established an important cytoprotective function in stress hematopoiesis with dichotomous megakaryocyte-biased hematopoietic recovery. Defective stress erythropoiesis was evident in Blvrb-/- spleens and in bone marrow erythroid development, occurring in conjunction with defective lipid peroxidation as a marker of oxidant mishandling. Cell autonomous effects on megakaryocyte lineage bias were documented using multipotential progenitor assays. These data provide the first physiological function of murine Blvrb in a non-redundant pathway of stress cytoprotection. Divergent effects on erythroid/megakaryocyte lineage speciation impute a novel redox-regulated mechanism for lineage partitioning.

Post-translational Succinylation of Mycobacterium tuberculosis Enoyl-CoA Hydratase EchA19 Slows Catalytic Hydration of Cholesterol Catabolite 3-Oxo-chol-4,22-diene-24-oyl-CoA.

Cholesterol is a major carbon source for Mycobacterium tuberculosis (Mtb) during infection, and cholesterol utilization plays a significant role in persistence and virulence within host macrophages. Elucidating the mechanism by which cholesterol is degraded may permit the identification of new therapeutic targets. Here, we characterized EchA19 (Rv3516), an enoyl-CoA hydratase involved in cholesterol side-chain catabolism. Steady-state kinetics assays demonstrated that EchA19 preferentially hydrates cholesterol enoyl-CoA metabolite 3-oxo-chol-4,22-diene-24-oyl-CoA, an intermediate of side-chain ß-oxidation. In addition, succinyl-CoA, a downstream catabolite of propionyl-CoA that forms during cholesterol degradation, covalently modifies targeted mycobacterial proteins, including EchA19. Inspection of a 1.9 Å resolution X-ray crystallography structure of Mtb EchA19 suggests that succinylation of Lys132 and Lys139 may perturb enzymatic activity by modifying the entrance to the substrate binding site. Treatment of EchA19 with succinyl-CoA revealed that these two residues are hotspots for succinylation. Replacement of these specific lysine residues with negatively charged glutamate reduced the rate of catalytic hydration of 3-oxo-chol-4,22-diene-24-oyl-CoA by EchA19, as does succinylation of EchA19. Our findings suggest that succinylation is a negative feedback regulator of cholesterol metabolism, thereby adding another layer of complexity to Mtb physiology in the host. These regulatory pathways are potential noncatabolic targets for antimicrobial drugs.

Biochemical characterization of biliverdins IXß/δ generated by a selective heme oxygenase.

The pro-oxidant effect of free heme (Fe2+-protoporphyrin IX) is neutralized by phylogenetically-conserved heme oxygenases (HMOX) that generate carbon monoxide, free ferrous iron, and biliverdin (BV) tetrapyrrole(s), with downstream BV reduction by non-redundant NADPH-dependent BV reductases (BLVRA and BLVRB) that retain isomer-restricted functional activity for bilirubin (BR) generation. Regioselectivity for the heme α-meso carbon resulting in predominant BV IXα generation is a defining characteristic of canonical HMOXs, thereby limiting generation and availability of BVs IXß, IXδ, and IXγ as BLVRB substrates. We have now exploited the unique capacity of the Pseudomonas aeruginosa (P. aeruginosa) hemO/pigA gene for focused generation of isomeric BVs (IXß and IXδ). A scalable system followed by isomeric separation yielded highly pure samples with predicted hydrogen-bonded structure(s) as documented by 1H NMR spectroscopy. Detailed kinetic studies established near-identical activity of BV IXß and BV IXδ as BLVRB-selective substrates, with confirmation of an ordered sequential mechanism of BR/NADP+ dissociation. Halogenated xanthene-based compounds previously identified as BLVRB-targeted flavin reductase inhibitors displayed comparable inhibition parameters using BV IXß as substrate, documenting common structural features of the cofactor/substrate-binding pocket. These data provide further insights into structure/activity mechanisms of isomeric BVs as BLVRB substrates, with potential applicability to further dissect redox-regulated functions in cytoprotection and hematopoiesis.

Heme degradation enzyme biliverdin IXß reductase is required for stem cell glutamine metabolism.

Bioenergetic requirements of hematopoietic stem cells and pluripotent stem cells (PSCs) vary with lineage fate, and cellular adaptations rely largely on substrate (glucose/glutamine) availability and mitochondrial function to balance tricarboxylic acid (TCA)-derived anabolic and redox-regulated antioxidant functions. Heme synthesis and degradation converge in a linear pathway that utilizes TCA cycle-derived carbon in cataplerotic reactions of tetrapyrrole biosynthesis, terminated by NAD(P)H-dependent biliverdin reductases (IXα, BLVRA and IXß, BLVRB) that lead to bilirubin generation and cellular antioxidant functions. We now demonstrate that PSCs with targeted deletion of BLVRB display physiologically defective antioxidant activity and cellular viability, associated with a glutamine-restricted defect in TCA entry that was computationally predicted using gene/metabolite topological network analysis and subsequently validated by bioenergetic and isotopomeric studies. Defective BLVRB-regulated glutamine utilization was accompanied by exaggerated glycolytic accumulation of the rate-limiting hexokinase reaction product glucose-6-phosphate. BLVRB-deficient embryoid body formation (a critical size parameter of early lineage fate potential) demonstrated enhanced sensitivity to the pentose phosphate pathway (PPP) inhibitor 6-aminonicotinamide with no differences in the glycolytic pathway inhibitor 2-deoxyglucose. These collective data place heme catabolism in a crucial pathway of glutamine-regulated bioenergetic metabolism and suggest that early stages of lineage fate potential require glutamine anaplerotic functions and an intact PPP, which are, in part, regulated by BLVRB activity. In principle, BLVRB inhibition represents an alternative strategy for modulating cellular glutamine utilization with consequences for cancer and hematopoietic metabolism.

In silico and crystallographic studies identify key structural features of biliverdin IXß reductase inhibitors having nanomolar potency.

Heme cytotoxicity is minimized by a two-step catabolic reaction that generates biliverdin (BV) and bilirubin (BR) tetrapyrroles. The second step is regulated by two non-redundant biliverdin reductases (IXα (BLVRA) and IXß (BLVRB)), which retain isomeric specificity and NAD(P)H-dependent redox coupling linked to BR's antioxidant function. Defective BLVRB enzymatic activity with antioxidant mishandling has been implicated in metabolic consequences of hematopoietic lineage fate and enhanced platelet counts in humans. We now outline an integrated platform of in silico and crystallographic studies for the identification of an initial class of compounds inhibiting BLVRB with potencies in the nanomolar range. We found that the most potent BLVRB inhibitors contain a tricyclic hydrocarbon core structure similar to the isoalloxazine ring of flavin mononucleotide and that both xanthene- and acridine-based compounds inhibit BLVRB's flavin and dichlorophenolindophenol (DCPIP) reductase functions. Crystallographic studies of ternary complexes with BLVRB-NADP+-xanthene-based compounds confirmed inhibitor binding adjacent to the cofactor nicotinamide and interactions with the Ser-111 side chain. This residue previously has been identified as critical for maintaining the enzymatic active site and cellular reductase functions in hematopoietic cells. Both acridine- and xanthene-based compounds caused selective and concentration-dependent loss of redox coupling in BLVRB-overexpressing promyelocytic HL-60 cells. These results provide promising chemical scaffolds for the development of enhanced BLVRB inhibitors and identify chemical probes to better dissect the role of biliverdins, alternative substrates, and BLVRB function in physiologically relevant cellular contexts.

Catabolism of the Cholesterol Side Chain in Mycobacterium tuberculosis Is Controlled by a Redox-Sensitive Thiol Switch.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a highly successful human pathogen and has infected approximately one-third of the world's population. Multiple drug resistant (MDR) and extensively drug resistant (XDR) TB strains and coinfection with HIV have increased the challenges of successfully treating this disease pandemic. The metabolism of host cholesterol by Mtb is an important factor for both its virulence and pathogenesis. In Mtb, the cholesterol side chain is degraded through multiple cycles of ß-oxidation and FadA5 (Rv3546) catalyzes side chain thiolysis in the first two cycles. Moreover, FadA5 is important during the chronic stage of infection in a mouse model of Mtb infection. Here, we report the redox control of FadA5 catalytic activity that results from reversible disulfide bond formation between Cys59-Cys91 and Cys93-Cys377. Cys93 is the thiolytic nucleophile, and Cys377 is the general acid catalyst for cleavage of the ß-keto-acyl-CoA substrate. The disulfide bond formed between the two catalytic residues Cys93 and Cys377 blocks catalysis. The formation of the disulfide bonds is accompanied by a large domain swap at the FadA5 dimer interface that serves to bring Cys93 and Cys377 in close proximity for disulfide bond formation. The catalytic activity of FadA5 has a midpoint potential of -220 mV, which is close to the Mtb mycothiol potential in the activated macrophage. The redox profile of FadA5 suggests that FadA5 is fully active when Mtb resides in the unactivated macrophage to maximize flux into cholesterol catabolism. Upon activation of the macrophage, the oxidative shift in the mycothiol potential will decrease the thiolytic activity by 50%. Thus, the FadA5 midpoint potential is poised to rapidly restrict cholesterol side chain degradation in response to oxidative stress from the host macrophage environment.

Enzymatic Activity and Thermodynamic Stability of Biliverdin IXß Reductase Are Maintained by an Active Site Serine.

Biliverdin reductase IXß (BLVRB) is a crucial enzyme in heme metabolism. Recent studies in humans have identified a loss-of-function mutation (Ser111Leu) that unmasks a fundamentally important role in hematopoiesis. We have undertaken experimental and thermodynamic modeling studies to provide further insight into the role of the cofactor in substrate accessibility and protein folding properties regulating BLVRB catalytic mechanisms. Site-directed mutagenesis with molecular dynamic (MD) simulations establish the critical role of NAD(P)H-dependent conformational changes on substrate accessibility by forming the "hydrophobic pocket", along with identification of a single key residue (Arg35) modulating NADPH/NADH selectivity. Loop80 and Loop120 block the hydrophobic substrate binding pocket in apo BLVRB (open), whereas movement of these structures after cofactor binding results in the "closed" (catalytically active) conformation. Both enzymatic activity and thermodynamic stability are affected by mutation(s) involving Ser111, which is located in the core of the BLVRB active site. This work 1) elucidates the crucial role of Ser111 in enzymatic catalysis and thermodynamic stability by active site hydrogen bond network; 2) defines a dynamic model for apo BLVRB extending beyond the crystal structure of the binary BLVRB/NADP+ complex; 3) provides a structural basis for the "encounter" and "equilibrium" states of the binary complex, which are regulated by NAD(P)H.

BLVRB redox mutation defines heme degradation in a metabolic pathway of enhanced thrombopoiesis in humans.

Human blood cell counts are tightly maintained within narrow physiologic ranges, largely controlled by cytokine-integrated signaling and transcriptional circuits that regulate multilineage hematopoietic specification. Known genetic loci influencing blood cell production account for <10% of platelet and red blood cell variability, and thrombopoietin/cellular myeloproliferative leukemia virus liganding is dispensable for definitive thrombopoiesis, establishing that fundamentally important modifier loci remain unelucidated. In this study, platelet transcriptome sequencing and extended thrombocytosis cohort analyses identified a single loss-of-function mutation (BLVRB(S111L)) causally associated with clonal and nonclonal disorders of enhanced platelet production. BLVRB(S111L) encompassed within the substrate/cofactor [α/ß dinucleotide NAD(P)H] binding fold is a functionally defective redox coupler using flavin and biliverdin (BV) IXß tetrapyrrole(s) and results in exaggerated reactive oxygen species accumulation as a putative metabolic signal leading to differential hematopoietic lineage commitment and enhanced thrombopoiesis. These data define the first physiologically relevant function of BLVRB and implicate its activity and/or heme-regulated BV tetrapyrrole(s) in a unique redox-regulated bioenergetic pathway governing terminal megakaryocytopoiesis; these observations also define a mechanistically restricted drug target retaining potential for enhancing human platelet counts.

Computer-aided identification, synthesis, and biological evaluation of novel inhibitors for botulinum neurotoxin serotype A.

Botulinum neurotoxins (BoNTs) are among the most potent biological toxin known to humans, and are classified as Category A bioterrorism agents by the Centers for Disease Control and prevention (CDC). There are seven known BoNT serotypes (A-G) which have been thus far identified in literature. BoNTs have been shown to block neurotransmitter release by cleaving proteins of the soluble NSF attachment protein receptor (SNARE) complex. Disruption of the SNARE complex precludes motor neuron failure which ultimately results in flaccid paralysis in humans and animals. Currently, there are no effective therapeutic treatments against the neurotoxin light chain (LC) after translocation into the cytosols of motor neurons. In this work, high-throughput in silico screening was employed to screen a library of commercially available compounds from ZINC database against BoNT/A-LC. Among the hit compounds from the in silico screening, two lead compounds were identified and found to have potent inhibitory activity against BoNT/A-LC in vitro, as well as in Neuro-2a cells. A few analogs of the lead compounds were synthesized and their potency examined. One of these analogs showed an enhanced activity than the lead compounds.

Modification of a bi-functional diguanylate cyclase-phosphodiesterase to efficiently produce cyclic diguanylate monophosphate.

Cyclic-diGMP is a bacterial messenger that regulates many physiological processes, including many attributed to pathogenicity. Bacteria synthesize cyclic-diGMP from GTP using diguanylate cyclases; its hydrolysis is catalyzed by phosphodiesterases. Here we report the over-expression and purification of a bi-functional diguanylate cyclase-phosphodiesterase from Agrobacterium vitis S4. Using homology modeling and primary structure alignment, we identify several amino acids predicted to participate in the phosphodiesterase reaction. Upon altering selected residues, we obtain variants of the enzyme that efficiently and quantitatively catalyze the synthesis of cyclic-diGMP from GTP without hydrolysis to pGpG. Additionally, we identify a variant that produces cyclic-diGMP while immobilized to NiNTA beads and can catalyze the conversion of [α-32P]-GTP to [32P]-cyclic-diGMP. In short, we characterize a novel cyclic-diGMP processing enzyme and demonstrate its utility for efficient and cost-effective production of cyclic-diGMP, as well as modified cyclic-diGMP molecules, for use as probes in studying the many important biological processes mediated by cyclic-diGMP.

FadA5 a thiolase from Mycobacterium tuberculosis: a steroid-binding pocket reveals the potential for drug development against tuberculosis.

With the exception of HIV, tuberculosis (TB) is the leading cause of mortality among infectious diseases. The urgent need to develop new antitubercular drugs is apparent due to the increasing number of drug-resistant Mycobacterium tuberculosis (Mtb) strains. Proteins involved in cholesterol import and metabolism have recently been discovered as potent targets against TB. FadA5, a thiolase from Mtb, is catalyzing the last step of the ß-oxidation reaction of the cholesterol side-chain degradation under release of critical metabolites and was shown to be of importance during the chronic stage of TB infections. To gain structural and mechanistic insight on FadA5, we characterized the enzyme in different stages of the cleavage reaction and with a steroid bound to the binding pocket. Structural comparisons to human thiolases revealed that it should be possible to target FadA5 specifically, and the steroid-bound structure provides a solid basis for the development of inhibitors against FadA5.

A thiolase of Mycobacterium tuberculosis is required for virulence and production of androstenedione and androstadienedione from cholesterol.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that shifts to a lipid-based metabolism in the host. Moreover, metabolism of the host lipid cholesterol plays an important role in M. tuberculosis infection. We used transcriptional profiling to identify genes transcriptionally regulated by cholesterol and KstR (Rv3574), a TetR-like repressor. The fadA5 (Rv3546) gene, annotated as a lipid-metabolizing thiolase, the expression of which is upregulated by cholesterol and repressed by KstR, was deleted in M. tuberculosis H37Rv. We demonstrated that fadA5 is required for utilization of cholesterol as a sole carbon source in vitro and for full virulence of M. tuberculosis in the chronic stage of mouse lung infection. Cholesterol is not toxic to the fadA5 mutant strain, and, therefore, toxicity does not account for its attenuation. We show that the wild-type strain, H37Rv, metabolizes cholesterol to androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD) and exports these metabolites into the medium, whereas the fadA5 mutant strain is defective for this activity. We demonstrate that FadA5 catalyzes the thiolysis of acetoacetyl-coenzyme A (CoA). This catalytic activity is consistent with a beta-ketoacyl-CoA thiolase function in cholesterol beta-oxidation that is required for the production of androsterones. We conclude that the attenuated phenotype of the fadA5 mutant is a consequence of disrupted cholesterol metabolism that is essential only in the persistent stage of M. tuberculosis infection and may be caused by the inability to produce AD/ADD from cholesterol.

Cholesterol metabolism increases the metabolic pool of propionate in Mycobacterium tuberculosis.

Mycobacterium tuberculosis can metabolize cholesterol to both acetate and propionate. The mass of isolated phthiocerol dimycoserate, a methyl-branched fatty acylated polyketide, was used as a reporter for intracellular propionate metabolic flux. When M. tuberculosis is grown using cholesterol as the only source of carbon, a 42 amu increase in average phthiocerol dimycoserate molecular weight is observed, consistent with the cellular pool of propionate and, thus, methylmalonyl CoA increasing upon cholesterol metabolism. In contrast, no shift in phthiocerol dimycoserate molecular weight is observed upon supplementation of medium containing glycerol and glucose with cholesterol. We conclude that cholesterol is a significant source of propionate only in the absence of sugar carbon sources.

Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase.

Lipoic acid is a sulfur-containing 8-carbon fatty acid that functions as a central cofactor in multienzyme complexes that are involved in the oxidative decarboxylation of glycine and several alpha-keto acids. In its functional form, it is bound covalently in an amide linkage to the epsilon-amino group of a conserved lysine residue of the "lipoyl bearing subunit," resulting in a long "swinging arm" that shuttles intermediates among the requisite active sites. In Escherichia coli and many other organisms, the lipoyl cofactor can be synthesized endogenously. The 8-carbon fatty-acyl chain is constructed via the type II fatty acid biosynthetic pathway as an appendage to the acyl carrier protein (ACP). Lipoyl(octanoyl)transferase (LipB) transfers the octanoyl chain from ACP to the target lysine acceptor, generating the substrate for lipoyl synthase (LS), which subsequently catalyzes insertion of both sulfur atoms into the C-6 and C-8 positions of the octanoyl chain. In this study, we present a three-step isolation procedure that results in a 14-fold purification of LipB to >95% homogeneity in an overall yield of 25%. We also show that the protein catalyzes the transfer of the octanoyl group from octanoyl-ACP to apo-H protein, which is the lipoyl bearing subunit of the glycine cleavage system. The specific activity of the purified protein is 0.541 U mg(-1), indicating a turnover number of approximately 0.2 s(-1), and the apparent Km values for octanoyl-ACP and apo-H protein are 10.2+/-4.4 and 13.2+/-2.9 microM, respectively.

Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide.

Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-l-methionine (SAM) as the precursor to a high-energy 5'-deoxyadenosyl 5'-radical (5'-dA(*)). In the LS reaction, the 5'-dA(*) is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d(15) H-protein of the glycine cleavage system-one of the substrates for LS-has been reported [Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Biochemistry 43, 6378-6386]. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X(3)-C-X(2)-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X(4)-C-X(5)-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 +/- 0.5 irons and 6.4 +/- 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5'-deoxyadenosine (5'-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 +/- 0.1 irons and 3.6 +/- 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 +/- 0.1 irons and 4.7 +/- 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5'-dA or the lipoyl group. Mössbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all associated iron is in the configuration [4Fe-4S](2+). When wild-type LS was reconstituted with (57)Fe and sodium sulfide, it harbored considerably more iron (13.8 +/- 0.6) and sulfide (13.1 +/- 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5'-dA and 0.36 equiv of the lipoyl group. Mössbauer spectroscopy of this protein revealed that only approximately 67% +/- 6% of the iron is in the form of [4Fe-4S](2+) clusters, amounting to 9.2 +/- 0.4 irons and 8.8 +/- 0.1 sulfides or 2 [4Fe-4S](2+) clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the Mössbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs.

Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid.

Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various alpha-keto acids, as well as the cleavage of glycine into CO(2) and NH(3), with concomitant transfer of its alpha-carbon to tetrahydrofolate, generating N(5),N(10)-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochemical studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-l-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA(*)), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming l-methionine in addition to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated derivative of the lipoyl-bearing subunit (E(2)) of the pyruvate dehydrogenase complex [Zhao, S., Miller, J. R., Jian, Y., Marletta, M. A., and Cronan, J. E., Jr. (2003) Chem. Biol. 10, 1293-1302]. Herein, we show that the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, we show that the 5'-dA(*) acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d(15)]H-protein to 5'-deoxyadenosine. Last, our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.