Lipoate Synthase Research Articles

Metabolic engineering of Escherichia coli for the production of (R)-α-lipoic acid.

To increase the production of (R)-α-lipoic acid directly from octanoic acid using engineered Escherichia coli with the regeneration of S-adenosylmethionine. The biosynthesis of (R)-α-lipoic acid (LA) in E. coli BL21(DE3) is improved by co-expression of lipoate-protein ligase A (LplA) from E. coli MG1655 and lipoate synthase (LipA) from Vibrio vulnificus. The engineered strain produces 20.99µgl-1 of LA in shake flask cultures. The titers of LA are increased to 169.28µgl-1 after the optimization of the medium components and fermentation conditions. We find that the [4Fe-4S] cluster is important for the activity of LipA and co-expression of iscSUA promotes the regeneration of the [4Fe-4S] cluster and leads to the highest LA titer of 589.30µgl-1. The method described here can be widely applied for the biosynthesis of (R)-α-lipoic acid and other metabolites.

Functional characterization of the first lipoyl-relay pathway from a parasitic protozoan.

Lipoic acid (LA) is a sulfur-containing cofactor covalently attached to key enzymes of central metabolism in prokaryotes and eukaryotes. LA can be acquired by scavenging, mediated by a lipoate ligase, or de novo synthesized by a pathway requiring an octanoyltransferase and a lipoate synthase. A more complex pathway, referred to as "lipoyl-relay", requires two additional proteins, GcvH, the glycine cleavage system H subunit, and an amidotransferase. This route was described so far in Bacillus subtilis and related Gram-positive bacteria, Saccharomyces cerevisiae, Homo sapiens, and Caenorhabditis elegans. Using collections of S. cerevisiae and B. subtilis mutants, defective in LA metabolism, we gathered evidence that allows us to propose for the first time that lipoyl-relay pathways are also present in parasitic protozoa. By a reverse genetic approach, we assigned octanoyltransferase and amidotransferase activity to the products of Tb927.11.9390 (TblipT) and Tb927.8.630 (TblipL) genes of Trypanosoma brucei, respectively. The B. subtilis model allowed us to identify the parasite amidotransferase as the target of lipoate analogs like 8-bromo-octanoic acid, explaining the complete loss of protein lipoylation and growth impairment caused by this compound in T. cruzi. This model could be instrumental for the screening of selective and more efficient chemotherapies against trypanosomiases.

Nonketotic hyperglycinemia in captive-bred Vervet monkeys (Chlorocebus aethiops) with cataracts.

Nonketotic hyperglycinemia (NKH) is a rare metabolic disorder that is characterized by high levels of glycine in plasma and cerebrospinal fluid in humans. In this study, total congenital cataract captive-bred Vervet monkeys (Chlorocebus aethiops) that are hyperglycinemic were screened to identify mutations in Bola type 3 (BOLA3), glutaredoxin 5 (GLRX5), and lipoate synthase (LIAS) genes. Twenty-four Vervet monkeys (12 hyperglycinemic and 12 healthy controls) were selected for mutation analysis using polymerase chain reaction (PCR), Sanger sequencing, and reverse transcriptase-polymerase chain reaction (RT-PCR). Novel sequence variants were identified in BOLA3 (R23H and Q38R) and LIAS (R369I and A371A), and gene expression in the control group was significantly lower compared to the hyperglycinemic group (P<0.05). The data obtained from this study will contribute to generation of new knowledge regarding the involvement of these genes in NKH development.

Characterization of a new N-terminally acetylated extra-mitochondrial isoform of frataxin in human erythrocytes

Frataxin is a highly conserved protein encoded by the frataxin (FXN) gene. The full-length 210-amino acid form of protein frataxin (1–210; isoform A) expressed in the cytosol of cells rapidly translocates to the mitochondria, where it is converted to the mature form (81–210) by mitochondrial processing peptidase. Mature frataxin (81–210) is a critically important protein because it facilitates the assembly of mitochondrial iron-sulfur cluster protein complexes such as aconitase, lipoate synthase, and succinate dehydrogenases. Decreased expression of frataxin protein is responsible for the devastating rare genetic disease of Friedreich’s ataxia. The mitochondrial form of frataxin has long been thought to be present in erythrocytes even though paradoxically, erythrocytes lack mitochondria. We have discovered that erythrocyte frataxin is in fact a novel isoform of frataxin (isoform E) with 135-amino acids and an N-terminally acetylated methionine residue. There is three times as much isoform E in erythrocytes (20.9 ± 6.4 ng/mL) from the whole blood of healthy volunteers (n = 10) when compared with the mature mitochondrial frataxin present in other blood cells (7.1 ± 1.0 ng/mL). Isoform E lacks a mitochondrial targeting sequence and so is distributed to both cytosol and the nucleus when expressed in cultured cells. When extra-mitochondrial frataxin isoform E is expressed in HEK 293 cells, it is converted to a shorter isoform identical to the mature frataxin found in mitochondria, which raises the possibility that it is involved in disease etiology. The ability to specifically quantify extra-mitochondrial and mitochondrial isoforms of frataxin in whole blood will make it possible to readily follow the natural history of diseases such as Friedreich’s ataxia and monitor the efficacy of therapeutic interventions.

Mitochondrial Bol1 and Bol3 function as assembly factors for specific iron-sulfur proteins

Assembly of mitochondrial iron-sulfur (Fe/S) proteins is a key process of cells, and defects cause many rare diseases. In the first phase of this pathway, ten Fe/S cluster (ISC) assembly components synthesize and insert [2Fe-2S] clusters. The second phase is dedicated to the assembly of [4Fe-4S] proteins, yet this part is poorly understood. Here, we characterize the BOLA family proteins Bol1 and Bol3 as specific mitochondrial ISC assembly factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. Bol1-Bol3 perform largely overlapping functions, yet cannot replace the ISC protein Nfu1 that also participates in this phase of Fe/S protein biogenesis. Bol1 and Bol3 form dimeric complexes with both monothiol glutaredoxin Grx5 and Nfu1. Complex formation differentially influences the stability of the Grx5-Bol-shared Fe/S clusters. Our findings provide the biochemical basis for explaining the pathological phenotypes of patients with mutations in BOLA3.

Protein–protein interaction studies provide evidence for electron transfer from ferredoxin to lipoic acid synthase in Toxoplasma gondii

The only known redox system in the apicoplast, a plastid-like organelle of apicomplexan parasites, is ferredoxin and ferredoxin-associated reductase. Ferredoxin donates electrons to different enzymes, presumably including lipoate synthase (LipA), which is essential for fatty acid biosynthesis. We recombinantly expressed and characterized LipA from the protozoan parasite Toxoplasma gondii, generated LipA-specific antibodies and confirmed the apicoplast localization of LipA. Electron transfer from ferredoxin to LipA would require direct protein–protein interaction. Such a robust interaction between the two proteins was demonstrated in both yeast and bacterial two-hybrid systems. Taken together, our results provide strong evidence for a role of ferredoxin as an electron donor to LipA.

Proteomic analysis reveals a novel function of the kinase Sat4p in Saccharomyces cerevisiae mitochondria.

The Saccharomyces cerevisiae kinase Sat4p has been originally identified as a protein involved in salt tolerance and stabilization of plasma membrane transporters, implicating a cytoplasmic localization. Our study revealed an additional mitochondrial (mt) localization, suggesting a dual function for Sat4p. While no mt related phenotype was observed in the absence of Sat4p, its overexpression resulted in significant changes of a specific mitochondrial subproteome. As shown by a comparative two dimensional difference gel electrophoresis (2D-DIGE) approach combined with mass spectrometry, particularly two groups of proteins were affected: the iron-sulfur containing aconitase-type proteins (Aco1p, Lys4p) and the lipoamide-containing subproteome (Lat1p, Kgd2p and Gcv3p). The lipoylation sites of all three proteins could be assigned by nanoLC-MS/MS to Lys75 (Lat1p), Lys114 (Kgd2p) and Lys102 (Gcv3p), respectively. Sat4p overexpression resulted in accumulation of the delipoylated protein variants and in reduced levels of aconitase-type proteins, accompanied by a decrease in the activities of the respective enzyme complexes. We propose a regulatory role of Sat4p in the late steps of the maturation of a specific subset of mitochondrial iron-sulfur cluster proteins, including Aco1p and lipoate synthase Lip5p. Impairment of the latter enzyme may account for the observed lipoylation defects.

Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5

Patients with nonketotic hyperglycinemia and deficient glycine cleavage enzyme activity, but without mutations in AMT, GLDC or GCSH, the genes encoding its constituent proteins, constitute a clinical group which we call 'variant nonketotic hyperglycinemia'. We hypothesize that in some patients the aetiology involves genetic mutations that result in a deficiency of the cofactor lipoate, and sequenced genes involved in lipoate synthesis and iron-sulphur cluster biogenesis. Of 11 individuals identified with variant nonketotic hyperglycinemia, we were able to determine the genetic aetiology in eight patients and delineate the clinical and biochemical phenotypes. Mutations were identified in the genes for lipoate synthase (LIAS), BolA type 3 (BOLA3), and a novel gene glutaredoxin 5 (GLRX5). Patients with GLRX5-associated variant nonketotic hyperglycinemia had normal development with childhood-onset spastic paraplegia, spinal lesion, and optic atrophy. Clinical features of BOLA3-associated variant nonketotic hyperglycinemia include severe neurodegeneration after a period of normal development. Additional features include leukodystrophy, cardiomyopathy and optic atrophy. Patients with lipoate synthase-deficient variant nonketotic hyperglycinemia varied in severity from mild static encephalopathy to Leigh disease and cortical involvement. All patients had high serum and borderline elevated cerebrospinal fluid glycine and cerebrospinal fluid:plasma glycine ratio, and deficient glycine cleavage enzyme activity. They had low pyruvate dehydrogenase enzyme activity but most did not have lactic acidosis. Patients were deficient in lipoylation of mitochondrial proteins. There were minimal and inconsistent changes in cellular iron handling, and respiratory chain activity was unaffected. Identified mutations were phylogenetically conserved, and transfection with native genes corrected the biochemical deficiency proving pathogenicity. Treatments of cells with lipoate and with mitochondrially-targeted lipoate were unsuccessful at correcting the deficiency. The recognition of variant nonketotic hyperglycinemia is important for physicians evaluating patients with abnormalities in glycine as this will affect the genetic causation and genetic counselling, and provide prognostic information on the expected phenotypic course.

Lipoic acid metabolism of Plasmodium--a suitable drug target.

α-Lipoic acid (6,8-thioctic acid; LA) is a vital co-factor of α-ketoacid dehydrogenase complexes and the glycine cleavage system. In recent years it was shown that biosynthesis and salvage of LA in Plasmodium are necessary for the parasites to complete their complex life cycle. LA salvage requires two lipoic acid protein ligases (LplA1 and LplA2). LplA1 is confined to the mitochondrion while LplA2 is located in both the mitochondrion and the apicoplast. LplA1 exclusively uses salvaged LA and lipoylates α-ketoglutarate dehydrogenase, branched chain α-ketoacid dehydrogenase and the H-protein of the glycine cleavage system. LplA2 cannot compensate for the loss of LplA1 function during blood stage development suggesting a specific function for LplA2 that has yet to be elucidated. LA salvage is essential for the intra-erythrocytic and liver stage development of Plasmodium and thus offers great potential for future drug or vaccine development.LA biosynthesis, comprising octanoyl-acyl carrier protein (ACP) : protein N-octanoyltransferase (LipB) and lipoate synthase (LipA), is exclusively found in the apicoplast of Plasmodium where it generates LA de novo from octanoyl-ACP, provided by the type II fatty acid biosynthesis (FAS II) pathway also present in the organelle. LA is the co-factor of the acetyltransferase subunit of the apicoplast located pyruvate dehydrogenase (PDH), which generates acetyl-CoA, feeding into FAS II. LA biosynthesis is not vital for intra-erythrocytic development of Plasmodium, but the deletion of several genes encoding components of FAS II or PDH was detrimental for liver stage development of the parasites indirectly suggesting that the same applies to LA biosynthesis.These data provide strong evidence that LA salvage and biosynthesis are vital for different stages of Plasmodium development and offer potential for drug and vaccine design against malaria.

Lipoic Acid Synthesis: A New Family of Octanoyltransferases Generally Annotated as Lipoate Protein Ligases

Bacillus subtilis lacks a recognizable homologue of the LipB octanoyltransferase, an enzyme essential for lipoic acid synthesis in Escherichia coli. LipB transfers the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the 2-oxoacid dehydrogenases via a thioester-linked octanoyl-LipB intermediate. The octanoylated dehydrogenase is then converted to the enzymatically active lipoylated species by insertion of two sulfur atoms into the octanoyl moiety by the S-adenosyl-l-methionine radical enzyme, LipA (lipoate synthase). B. subtilis synthesizes lipoic acid and contains a LipA homologue that is fully functional in E. coli. Therefore, the lack of a LipB homologue presented the puzzle of how B. subtilis synthesizes the LipA substrate. We report that B. subtilis encodes an octanoyltransferase that has virtually no sequence resemblance to E. coli LipB but instead has a sequence that resembles that of the E. coli lipoate ligase, LplA. On the basis of this resemblance, these genes have generally been annotated as encoding a lipoate ligase, an enzyme that in E. coli scavenges lipoic acid from the environment but plays no role in de novo synthesis. We have named the B. subtilis octanoyltransferase LipM and find that, like LipB, the LipM reaction proceeds through a thioester-linked acyl enzyme intermediate. The LipM active site nucleophile was identified as C150 by the finding that this thiol becomes modified when LipM is expressed in E. coli. The level of the octanoyl-LipM intermediate can be significantly decreased by blocking fatty acid synthesis during LipM expression, and C150 was confirmed as an essential active site residue by site-directed mutagenesis. LipM homologues seem the sole type of octanoyltransferase present in the firmicutes and are also present in the cyanobacteria. LipM type octanoyltransferases represent a new clade of the PF03099 protein family, suggesting that octanoyl transfer activity has evolved at least twice within this superfamily.

Chlamydia trachomatisSerovar L2 Can Utilize Exogenous Lipoic Acid through the Action of the Lipoic Acid Ligase LplA1

Lipoic acid is an essential protein bound cofactor that is vital for the functioning of several important enzymes involved in central metabolism. Genomes of all sequenced chlamydiae show the presence of two genes encoding lipoic acid ligases and one gene encoding a lipoate synthase. However, the roles of these proteins in lipoic acid utilization or biosynthesis have not yet been characterized. The two distinct lipoic acid ligases in Chlamydia trachomatis serovar L2, LplA1(Ct) and LplA2(Ct) (encoded by the open reading frames ctl0537 and ctl0761) display moderate identity with Escherichia coli LplA (30 and 27%, respectively) but possess amino acid sequence motifs that are well conserved among all lipoyl protein ligases. The putative lipoic acid synthase LipA(Ct), encoded by ctl0815, is ca. 43% identical to the E. coli LipA homolog. We demonstrate here the presence of lipoylated proteins in C. trachomatis serovar L2 and show that the lipoic acid ligase LplA1(Ct) is capable of utilizing exogenous lipoic acid for the lipoylation Therefore, host-derived lipoic acid may be important for intracellular growth and development. Based on genetic complementation in a surrogate host, our study also suggests that the C. trachomatis serovar L2 LipA homolog may not be functional in vivo.

Integrative inference of gene-regulatory networks in Escherichia coli using information theoretic concepts and sequence analysis

BackgroundAlthough Escherichia coli is one of the best studied model organisms, a comprehensive understanding of its gene regulation is not yet achieved. There exist many approaches to reconstruct regulatory interaction networks from gene expression experiments. Mutual information based approaches are most useful for large-scale network inference.ResultsWe used a three-step approach in which we combined gene regulatory network inference based on directed information (DTI) and sequence analysis. DTI values were calculated on a set of gene expression profiles from 19 time course experiments extracted from the Many Microbes Microarray Database. Focusing on influences between pairs of genes in which one partner encodes a transcription factor (TF) we derived a network which contains 878 TF - gene interactions of which 166 are known according to RegulonDB. Afterward, we selected a subset of 109 interactions that could be confirmed by the presence of a phylogenetically conserved binding site of the respective regulator. By this second step, the fraction of known interactions increased from 19% to 60%. In the last step, we checked the 44 of the 109 interactions not yet included in RegulonDB for functional relationships between the regulator and the target and, thus, obtained ten TF - target gene interactions. Five of them concern the regulator LexA and have already been reported in the literature. The remaining five influences describe regulations by Fis (with two novel targets), PhdR, PhoP, and KdgR. For the validation of our approach, one of them, the regulation of lipoate synthase (LipA) by the pyruvate-sensing pyruvate dehydrogenate repressor (PdhR), was experimentally checked and confirmed.ConclusionsWe predicted a set of five novel TF - target gene interactions in E. coli. One of them, the regulation of lipA by the transcriptional regulator PdhR was validated experimentally. Furthermore, we developed DTInfer, a new R-package for the inference of gene-regulatory networks from microarrays using directed information.

Erythropoiesis and Iron Sulfur Cluster Biogenesis

Erythropoiesis in animals is a synchronized process of erythroid cell differentiation that depends on successful acquisition of iron. Heme synthesis depends on iron through its dependence on iron sulfur (Fe-S) cluster biogenesis. Here, we review the relationship between Fe-S biogenesis and heme synthesis in erythropoiesis, with emphasis on the proteins, GLRX5, ABCB7, ISCA, and C1orf69. These Fe-S biosynthesis proteins are highly expressed in erythroid tissues, and deficiency of each of these proteins has been shown to cause anemia in zebrafish model. GLRX5 is involved in the production and ABCB7 in the export of an unknown factor that may function as a gauge of mitochondrial iron status, which may indirectly modulate activity of iron regulatory proteins (IRPs). ALAS2, the enzyme catalyzing the first step in heme synthesis, is translationally controlled by IRPs. GLRX5 may also provide Fe-S cofactor for ferrochelatase, the last enzyme in heme synthesis. ISCA and C1orf69 are thought to assemble Fe-S clusters for mitochondrial aconitase and for lipoate synthase, the enzyme producing lipoate for pyruvate dehydrogenase complex (PDC). PDC and aconitase are involved in the production of succinyl-CoA, a substrate for heme biosynthesis. Thus, many steps of heme synthesis depend on Fe-S cluster assembly.

Chaperone-aided expression of LipA and LplA followed by the increase in α-lipoic acid production

Alpha-lipoic acid (LA), a naturally occurring cofactor reported to be present in a diverse group of microorganisms, plants, and animal tissues, has been widely and successfully used as a therapy for a variety of diseases, including diabetes and heart disease. However, to date, recombinant DNA technology has not been applied for higher LA production due mainly to difficulties in the functional expression of key enzymes involved in LA production. Here, we report a study for higher LA production with the aid of chaperone plasmids, DnaKJE and trigger factor (Tf). The lipA and lplA genes encoding lipoate synthase and lipoate protein ligase in Pseudomonas fluorescens, respectively, were cloned and transformed into Escherichia coli K12. When they were overexpressed in E. coli, both LipA and LplA were expressed as inclusion bodies leading to no increase in LA production. However, when chaperone plasmids DnaKJE and Tf were coexpressed with lipA and lplA, the resulting recombinant E. coli strains showed higher LA production than the wild-type E. coli by 32-111%, respectively.

A newly discovered role for iron-sulfur clusters

Nature has exploited the structural and electronic plasticity of small inorganic structures called [Fe-S] clusters (Fig. 1) in at least five fundamental ways: (i) electron transfer, (ii) Lewis acid-assisted enzyme catalysis, (iii) radical generation, (iv) sulfur donation, and (v) control of protein conformational changes associated with signal transduction (1). In their role as agents of electron transfer, it is known that polypeptide environments that surround [Fe-S] clusters modulate their midpoint potentials so that electron flow can be controlled and directed. In their capacity to function as a Lewis acid, [Fe-S] clusters facilitate activation of a variety of electron-rich organic substrates. The dehydratase reaction within the tricarboxylic acid cycle, catalyzed by aconitase, provides one example of this type of [Fe-S] cluster-assisted metabolic transformation. For radical-mediated catalysis, [Fe-S] clusters interact with S-adenosylmethione to initiate formation of an activated adenosyl radical, which is subsequently used for generation of protein- or substrate-associated radical intermediates. Members of this class of enzyme include anaerobic ribonucleotide reductase, biotin synthase, and lipoate synthase. Some members of the S-adenosylmethione family of [Fe-S] proteins also appear to use an additional “sacrificial” [Fe-S] cluster as the source of S necessary for the assembly of certain cofactors such as biotin and lipoic acid. The facile assembly, disassembly, and rearrangement of [Fe-S] clusters in response to Fe or oxygen availability, or when exposed to superoxide, has resulted in their ability to serve as environmental sensors. For example, the activity of regulatory proteins that contain [Fe-S] clusters is frequently controlled through conformational changes affected by the redox state or nuclearity of their cognate [Fe-S] clusters. In this issue of PNAS, Luis Rubio, David Britt, and their colleagues (2) propose yet another function for an [Fe-S] cluster. In this case, the cluster is involved in the sequestration and delivery of Mo required for …

MiaB, a Bifunctional Radical-S-Adenosylmethionine Enzyme Involved in the Thiolation and Methylation of tRNA, Contains Two Essential [4Fe-4S] Clusters

The radical-S-adenosylmethionine (radical-AdoMet) enzyme MiaB catalyzes the posttranscriptional methylthiolation of N-6-isopentenyladenosine in tRNAs. Spectroscopic and analytical studies of the reconstituted wild-type and C150/154/157A triple variant forms of Thermotoga maritima MiaB have revealed the presence of two distinct [4Fe-4S](2+,1+) clusters in the protein. One is coordinated by the three conserved cysteines in the radical-AdoMet motif (Cys150, Cys154, and Cys157) as previously reported, and the other, here observed for the first time, is proposed to be coordinated by the three N-terminal conserved cysteines (Cys10, Cys46, and Cys79). The two [4Fe-4S]2+ clusters have similar UV-visible absorption, resonance Raman, and Mössbauer properties but differ in terms of redox properties and the EPR properties of the reduced [4Fe-4S]1+ clusters. Reconstituted forms of MiaB containing two [4Fe-4S] clusters are more active than previously reported. Comparison of MiaB with other radical-AdoMet enzymes involved in thiolation reactions, such as biotin synthase and lipoate synthase, is discussed as well as a possible role of the second cluster as a sacrificial S-donor in the MiaB-catalyzed reaction.

Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast

In contrast to other eukaryotes, which manufacture lipoic acid, an essential cofactor for several vital dehydrogenase complexes, within the mitochondrion, we show that the plastid (apicoplast) of the obligate intracellular protozoan parasite Toxoplasma gondii is the only site of de novo lipoate synthesis. However, antibodies specific for protein-attached lipoate reveal the presence of lipoylated proteins in both, the apicoplast and the mitochondrion of T. gondii. Cultivation of T. gondii-infected cells in lipoate-deficient medium results in substantially reduced lipoylation of mitochondrial (but not apicoplast) proteins. Addition of exogenous lipoate to the medium can rescue this effect, showing that the parasite scavenges this cofactor from the host. Exposure of T. gondii to lipoate analogues in lipoate-deficient medium leads to growth inhibition, suggesting that T. gondii might be auxotrophic for this cofactor. Phylogenetic analyses reveal the secondary loss of the mitochondrial lipoate synthase gene after the acquisition of the plastid. Our studies thus reveal an unexpected metabolic deficiency in T. gondii and raise the question whether the close interaction of host mitochondria with the parasitophorous vacuole is connected to lipoate supply by the host.

MiaB Protein from Thermotoga maritima: CHARACTERIZATION OF AN EXTREMELY THERMOPHILIC tRNA-METHYLTHIOTRANSFERASE

In Escherichia coli, the MiaB protein catalyzes the methylthiolation of N-6-isopentenyl adenosine in tRNAs, the last reaction step during biosynthesis of 2-methylthio-N-6-isopentenyl adenosine (ms2i6A-37). For the first time the thermophilic bacterium Thermotoga maritima is shown here to contain such a MiaB tRNA-modifying enzyme, named MiaBTm, and to synthesize ms2i6A-37 as demonstrated by an analysis of modified nucleosides from tRNA hydrolysates. The corresponding gene (TM0653) was identified by sequence similarity to the miaB gene cloned and expressed in E. coli. MiaBTm was purified to homogeneity and thoroughly characterized by biochemical and spectroscopic methods. It is a monomer of 443 residues with a molecular mass of 50,710 kilodaltons. Its amino acid sequence shares the CysXXX-CysXXCys sequence with MiaB from E. coli as well as with biotin synthase and lipoate synthase. This sequence was shown to be essential for chelation of an iron-sulfur center and for activity in these enzymes. As isolated, MiaBTm contains both iron and sulfide and an apoprotein form can coordinate up to 4 iron and 4 sulfur atoms per polypeptide chain. UV-visible absorption, resonance Raman, variable temperature magnetic circular dichroism, and EPR spectroscopy of MiaBTm indicate the presence of a [4Fe-4S]+2/+1 cluster under reducing and anaerobic conditions, whereas [3Fe-4S]+1 and [2Fe-2S]+2 forms are generated under aerobic conditions. The redox potential of the [4Fe-4S]+2/+1 transition is -495 +/- 10 mV (versus the normal hydrogen electrode). Finally, the expression of MiaBTm from T. maritima in an E. coli mutant strain lacking functional miaB gene allowed production of ms2i6A-37. These results provide further information on the enzymes involved in methylthiolation of tRNAs.

Radical mechanisms of enzymatic catalysis.

Two classes of enzymatic mechanisms that proceed by free radical chemistry initiated by the 5'-deoxyadenosyl radical are discussed. In the first class, the mechanism of the interconversion of L-lysine and L-beta-lysine catalyzed by lysine 2,3-aminomutase (LAM) involves four radicals, three of which have been spectroscopically characterized. The reversible formation of the 5'-deoxyadenosyl radical takes place by the chemical cleavage of S-adenosylmethionine (SAM) reacting with the [4Fe-4S]+ center in LAM. In other reactions of SAM with iron-sulfur proteins, SAM is irreversibly consumed to generate the 5'-deoxyadenosyl radical, which activates an enzyme by abstracting a hydrogen atom from an enzymatic glycyl residue to form a glycyl radical. The glycyl radical enzymes include pyruvate formate-lyase, anaerobic ribonucleotide reductase from Escherichia coli, and benzylsuccinate synthase. Biotin synthase and lipoate synthase are SAM-dependent [4Fe-4S] proteins that catalyze the insertion of sulfur into unactivated C-H bonds, which are cleaved by the 5'-deoxyadenosyl radical from SAM. In the second class of enzymatic mechanisms using free radicals, adenosylcobalamin-dependent reactions, the 5'-deoxyadenosyl radical arises from homolytic cleavage of the cobalt-carbon bond, and it initiates radical reactions by abstracting hydrogen atoms from substrates. Three examples are described of suicide inactivation through the formation of exceptionally stable free radicals at enzymatic active sites.

Biosynthesis of biotin and lipoic acid

The genetics and mechanistic enzymology of biotin biosynthesis have been the subject of much investigation in the last decade, owing to the interest for biotin production by fermentation, on the one hand, and for the design of inhibitors with potential herbicidal properties, on the other hand. Four enzymes are involved in the synthesis of biotin from its two precursors, alanine and pimeloyl-CoA. They are now well-characterized and the X-ray structures of the first three have been published. 8-Amino-7-oxopelargonic acid synthase is a pyridoxal 5'-phosphate (PLP) enzyme, very similar to other acyl-CoA alpha-oxoamine synthases, and its detailed mechanism has been determined. The origin of its specific substrate, pimeloyl-CoA, however, is not completely established. It could be produced by a modified fatty acid pathway involving a malonyl thioester as the starter. 7,8-Diaminopelargonic acid (DAPA) aminotransferase, although sharing sequence and folding homologies with other transaminases, is unique as it uses S-adenosylmethionine (AdoMet) as the NH2 donor. The mechanism of dethiobiotin synthethase is also now well understood. It catalyzes the formation of the ureido ring via a DAPA carbamate activated with ATP. On the other hand, the mechanism of the last enzyme, biotin synthase, which has long raised a very puzzling problem, is only starting to be unraveled and appears indeed to be very complex. Biotin synthase belongs to the family of AdoMet-dependent enzymes that reductively cleave AdoMet into a deoxyadenosyl radical, and it is responsible for the homolytic cleavage of C-H bonds. A first radical formed on dethiobiotin is trapped by the sulfur donor, which was found to be the iron-sulfur (Fe-S) center contained in the enzyme, and cyclization follows in a second step. Two important features come from these results: (1) a new role for an Fe-S center has been revealed, and (2) biotin synthase is not only a catalyst but also a substrate for the reaction. Lipoate synthase, which catalyzes the formation of two C-S bonds from octanoic acid, has a very high sequence similarity with biotin synthase. Although no in vitro enzymology has been carried out with lipoate synthase, the sequence homology as well as the results of in vivo studies support the conclusion that both enzymes are strongly mechanistically related.