Lipoate metabolism in Pseudomonas putida LP

dl-[1,6-¹⁴C]Lipoate was used to support the growth of Pseudomonas putida LP, which was found to grow on d- or l-lipoate as sole source of carbon and sulfur. The major radioactive catabolite in the benzene extract from acidified aerobic cultures was identified to be bisnorlipoate. The principal acidic ¹⁴C-catabolites in the aqueous phase have now been isolated and identified as β-hydroxybisnorlipoate, as well as bisnorlipoate; the existence of lesser amounts of tetranorlipoate is also indicated by Chromatographic evidence. Although the microorganism can grow on 8-methyllipoate (6,8-dithiononanoate), the bisnor- and tetranor-compounds, as well as 6,9-dithiononanoate (a dithiane derivative), do not support growth. Hence, the bacterium can derive most of the needed carbon by β-oxidation of the acid side chain of a 3-substituted dithiolane to yield the two-carbon-shorter bisnor-compound. Less extensive degradation of bisnorlipoate results in the formation of β-hydroxybisnorlipoate, which may be further metabolized to tetranorlipoate.     

Lipoic acid metabolism in the rat

dl-[1,6-¹⁴C]Lipoic acid was administered by intraperitoneal injection to rats at the level of 0.5 mg/100 g body weight. Approximately 56% of the radioactivity was recovered in the urine. When acidified and extracted with benzene, 92% of the radioactivity remained in the aqueous phase. Gel-filtration and paper chromatography were used to identify three of the compounds in the benzene extract as lipoic, bisnorlipoic and tetranorlipoic acids. In addition, a keto compound appears to be present. The aqueous phase contained several radioactive components separable by ion-exchange and paper chromatographies. Two of these compounds were identified as lipoate and β-hydroxybisnorlipoate. No evidence for oxidation of the dithiolane ring of lipoic acid was observed. dl-[7,8-¹⁴C]Lipoic acid was administered to rats under the same conditions. The urine contained 81% of the radioactivity, 72% of which remained in the aqueous phase and 28% was extracted into benzene. In contrast to over 30% of the label from dl-(1,6-¹⁴C] lipoate being expired as ¹⁴CO₂, a negligible amount of ¹⁴CO₂ was produced by rats injected with dl-[7,8-¹⁴C]lipoate. The catabolites identified were the same as those found using the 1,6-labeled lipoate. Another dithiolane-intact compound was also isolated. It appears that the rat, similar to Pseudomonas putida LP, metabolizes lipoate mainly via β-oxidation of the valeric acid side chain.     

Molecular cloning of cDNA for rat liver lipoate acetyltransferase a component of pyruvate dehydrogenase complex

One cDNA clone for lipoate acetyltransferase, a component enzyme of pyruvate dehydrogenase complex, was isolated from a rat liver cDNA library prepared in the phage expression vector λgt11 using immunological screening with affinity purified anti-lipoate acetyltransferase antibody. It was identified that cDNA insert in this clone codes for lipoate acetyltransferase by immunoblotting of lysogen carrying the isolated clone. Lipoate acetyltransferase antigenic polypeptide in fusion protein was about 11,000 daltons, agreeing with the size of cDNA insert to be 300 base pairs.     

Unravelling the lipoyl‐relay of exogenous lipoate utilization in Bacillus subtilis

Lipoate is an essential cofactor for key enzymes of oxidative and one‐carbon metabolism. It is covalently attached to E2 subunits of dehydrogenase complexes and GcvH, the H subunit of the glycine cleavage system. Bacillus subtilis possess two protein lipoylation pathways: biosynthesis and scavenging. The former requires octanoylation of GcvH, insertion of sulfur atoms and amidotransfer of the lipoate to E2s, catalyzed by LipL. Lipoate scavenging is mediated by a lipoyl protein ligase (LplJ) that catalyzes a classical two‐step ATP‐dependent reaction. Although these pathways were thought to be redundant, a ?lipL mutant, in which the endogenous lipoylation pathway of E2 subunits is blocked, showed growth defects in minimal media even when supplemented with lipoate and despite the presence of a functional LplJ. In this study, we demonstrate that LipL is essential to modify E2 subunits of branched chain ketoacid and pyruvate dehydrogenases during lipoate scavenging. The crucial role of LipL during lipoate utilization relies on the strict substrate specificity of LplJ, determined by charge complementarity between the ligase and the lipoylable subunits. This new lipoyl‐relay required for lipoate scavenging highlights the relevance of the amidotransferase as a valid target for the design of new antimicrobial agents among Gram‐positive pathogens.     

Stability and structure of binary and ternary complexes of alpha-lipoate and lipoate derivatives with Mn2+, Cu2+, and Zn2+ in solution.

The stabilities of the 1:1 complexes of Mn²⁺, Cu²⁺, and Zn²⁺ with lipoate and its chainshortened catabolites, viz., bisnorlipoate and tetranorlipoate, were studied by potentiometric titrations in water containing 50% dioxane (I = 0.1, NaClO₄; 25 °C). A comparison of the stabilities of these complexes with those of simple carboxylates reveals that the catabolite complexes formed with Cu²⁺ and Zn²⁺ are more stable than expected from only the basicity of the carboxylate groups. This is evidence that chelates involving the disulfide group are formed. The stability of all Mn²⁺ complexes is determined by the basicity of the carboxylate groups. The same pattern of stability holds for the mixed-ligand complexes formed by Cu²⁺ or Zn²⁺, 2,2′-bipyridyl, and lipoate or one of its derivatives. It is evident that the disulfide group of the 1,2-dithiolane moiety can participate in the formation of binary and ternary complexes. The somewhat less-pronounced coordinating properties of the 1,2-dithiolane moiety compared with the tetrahydrothiophene moiety are discussed. It is apparent that the electron density at S(1) and S(2) in the dithiolane moiety of lipoate is not equivalent: S(1) is favored over S(2) in electrophilic reactions; possible biological implications are indicated.     

Global Conformational Change Associated with the Two-step Reaction Catalyzed by Escherichia coli Lipoate-Protein Ligase A

Lipoate-protein ligase A (LplA) catalyzes the attachment of lipoic acid to lipoate-dependent enzymes by a two-step reaction: first the lipoate adenylation reaction and, second, the lipoate transfer reaction. We previously determined the crystal structure of Escherichia coli LplA in its unliganded form and a binary complex with lipoic acid (Fujiwara, K., Toma, S., Okamura-Ikeda, K., Motokawa, Y., Nakagawa, A., and Taniguchi, H. (2005) J Biol. Chem. 280, 33645–33651). Here, we report two new LplA structures, LplA·lipoyl-5′-AMP and LplA·octyl-5′-AMP·apoH-protein complexes, which represent the post-lipoate adenylation intermediate state and the pre-lipoate transfer intermediate state, respectively. These structures demonstrate three large scale conformational changes upon completion of the lipoate adenylation reaction: movements of the adenylate-binding and lipoate-binding loops to maintain the lipoyl-5′-AMP reaction intermediate and rotation of the C-terminal domain by about 180°. These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys¹³³ residue plays essential roles in both lipoate adenylation and lipoate transfer reactions. Based on structural and kinetic data, we propose a reaction mechanism driven by conformational changes.     

Hepatic lipoate uptake

Uptake of [35S]lipoate was studied in perfused rat liver and in isolated rat hepatocytes. During single-pass perfusion of [35S]lipoate about 30% of the radioactivity is retained in the liver. A substantial amount of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive material appears in the effluent perfusate, while hepatic efflux of GSH is unchanged. The hepatic uptake of lipoate, the release of thiols, and also the biliary excretion of 35S-labeled compounds are suppressed by octanoate. In isolated hepatocytes the uptake of lipoate follows saturation kinetics showing a Km value of 38 microM and a Vmax of 180 pmol/mg X 10 s. The uptake is temperature-dependent; from the Arrhenius plot an activation energy of 14.8 kcal/mol at 20 microM lipoate is calculated. At high concentrations of lipoate (above 75 microM) a nonsaturable uptake component becomes predominant. Lipoate uptake is selectively inhibited by medium-chain fatty acids. Only slight inhibition is seen in the presence of long-chain fatty acids, and there is no inhibition with acetate or lactate. Substantial inhibition is also observed with acetylsalicylic acid, but not with taurocholate, bromosulfophthalein or biotin. Lipoate uptake can be inhibited by high concentrations of phloretin (200 microM) and is rather insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (200 microM). The results indicate that hepatic uptake of lipoate at physiological concentrations is largely carrier-mediated.     

Studies on the efficacy of lipoate and dihydrolipoate in the alteration of cadmium2+ toxicity in isolated hepatocytes

Lipoate (thioctic acid) is presently used in therapy of a variety of diseases such as liver and neurological disorders. However, nothing is known about the efficacy of lipoate and its reduced form dihydrolipoate in acute cadmium (Cd2+) toxicity which involves severe liver disturbances. Therefore, we investigated the effects of these redox compounds on Cd2(+)-induced injuries in isolated rat hepatocytes. The cells were coincubated with 150 microM Cd2+ and either 1.5-6.0 mM lipoate or 17-89 microM dihydrolipoate for up to 90 min and Cd2+ uptake as well as viability criteria were monitored. Both exposure regimens diminished Cd2+ uptake in correspondence to time and concentration. They also ameliorated Cd2(+)-induced cell deterioration as reflected by the decrease in Cd2(+)-induced membrane damage (leakage of aspartate aminotransferase), by the lessening of the Cd2(+)-stimulated lipid peroxidation (TBA-reactants) and by the increase in Cd2(+)-depleted cellular glutathione (GSH + 2 GSSG). Half-maximal protection was achieved at molar ratios of 9.9 to 19 (lipoate vs. Cd2+) and 0.25 to 0.74 (dihydrolipoate vs. Cd2+), indicating a 19.5 to 50.6 lower protective efficacy of lipoate as compared to dihydrolipoate. Lipoate induced an increase in extracellular acid-soluble thiols different from glutathione. It is suggested that dihydrolipoate primarily protects cells by extracellular chelation of Cd2+, whereas intracellular reduction of lipoate to the dihydro-compound followed by complexation of both intra- and extracellular Cd2+ contributes to the amelioration provided by lipoate.     

Nutritional growth requirements forButyribacterium methylotrophicum on single carbon substrates and glucose

Butyribacterium methylotrophicum, an anaerobic acetogen, obligately required pantothenate for growth on either glucose, CH₃OH?CO₂, H₂?CO₂, or carbon monoxide. Growth on glucose but not single carbon substrates was stimulated by lipoate and biotin. Sulfide but not sulfate served as the sole sulfur source for growth. This study established thatB. methylotrophicum was both a true autotroph when grown on H₂?CO₂ and a unicarbonotroph on CO as the sole carbon and energy source. In addition, the vitamin requirements of this species further suggest its distinctiveness fromEubacterium limosum (Butyribacterium rettgeri).     

Synthesis of the metabolites of a free radical scavenger edaravone (MCI-186, Radicut™)

Abstract

Two metabolites of a free radical scavenger, edaravone, were synthesized. Edaravone glucuronate was synthesized by glycosylation of a glucuronic acid precursor using silver (I) trifluoromethane-sulfonate with edaravone. Edaravone sulfate was synthesized by sulfonylation of edaravone using a sulfur trioxide-pyridine complex. The two synthesized metabolites were identical to isolated metabolites. X-ray analysis identified edaravone glucuronate as β-O-glucuronate, although there were three possible edaravone glucuronate tautomers.     

The Streptomyces coelicolor Lipoate-protein Ligase Is a Circularly Permuted Version of the Escherichia coli Enzyme Composed of Discrete Interacting Domains

Lipoate-protein ligases are used to scavenge lipoic acid from the environment and attach the coenzyme to its cognate proteins, which are generally the E2 components of the 2-oxoacid dehydrogenases. The enzymes use ATP to activate lipoate to its adenylate, lipoyl-AMP, which remains tightly bound in the active site. This mixed anhydride is attacked by the ϵ-amino group of a specific lysine present on a highly conserved acceptor protein domain, resulting in the amide-linked coenzyme. The Streptomyces coelicolor genome encodes only a single putative lipoate ligase. However, this protein had only low sequence identity (<25%) to the lipoate ligases of demonstrated activity and appears to be a circularly permuted version of the known lipoate ligase proteins in that the canonical C-terminal domain seems to have been transposed to the N terminus. We tested the activity of this protein both by in vivo complementation of an Escherichia coli ligase-deficient strain and by in vitro assays. Moreover, when the domains were rearranged into a protein that mimicked the arrangement found in the canonical lipoate ligases, the enzyme retained complementation activity. Finally, when the two domains were separated into two proteins, both domain-containing proteins were required for complementation and catalysis of the overall ligase reaction in vitro. However, only the large domain-containing protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins, whereas both domain-containing proteins were required to form lipoyl-AMP.     

Purification properties and subunit composition of pig heart lipoate acetyltransferase.

Lipoate acetyltransferase [acetyl-CoA: dihydrolipoate S-acetyl-transferase, EC 2.3.1.12], the core enzyme of the pyruvate dehydrogenase complex, has been highly purified by gel chromatography on Sepharose 6B and sucrose density gradient centrifugation in the presence of potassium iodide. The native enzyme has a sedimentation coefficient (S020,W) of 26.7S and a diffusion coefficient (D020,W) of 1.25 x 10(-7) cm2.-sec-1. The weight-average molecular weight was estimated to be 1.8 million from the sedimentation equilibrium data. The content of right-handed alpha helix in the enzyme molecule was estimated to be about 25% by optical rotatory dispersion and about 22% from the circular dichroism spectra. The enzyme was found to contain about 23 moles of protein-bound lipoic acid per mole of enzyme; some other properties are also reported. Lipoate acetyltransferase dissociated to yield a single subunit with a molecular weight of 74,000 as estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by gel filtration on Bio-Gel in 6 M guanidine-HCl. The molecular weight was also estimated to be 74,000 from sedimentation equilibrium data in 6 M guanidine-HCl] containing 0.1 M 2-mercaptoethanol. Evidence is presented that 1 molecule of lipoate acetyltransferase apparently consists of 24 very similar subunits, each of which contains NH2-terminal alanine. Each subunit contains 1 molecule of covalently bound lipoic acid.     

The lipoate synthase from Escherichia coli is an iron-sulfur protein.

Lipoate synthase catalyzes the last step of the biosynthesis of lipoic acid in microorganisms and plants. The protein isolated from an overexpressing Escherichia coli strain was purified from inclusion bodies. Spectroscopic (UV-visible and electron paramagnetic resonance) properties of the reconstituted protein demonstrate the presence of a (2Fe-2S) center per protein. As observed in biotin synthase, these clusters are converted to (4Fe-4S) centers during reduction under anaerobic conditions. The possible involvement of the cluster in the insertion of sulfur atoms into the octanoic acid backbone is discussed.     

The metabolism of dl-(1,6-14C)lipoic acid in the rat

dl-[1,6-¹⁴C]Lipoic acid was synthesized and administered to rats or incubated in vitro with rat liver systems. The urinary excretion of radioactivity after labeled lipoate was administered intraperitoneally at a level of 0.5 mg/100 g body weight was maximal at 3–6 hr, with 60% of the injected radioactivity recovered within 24 hr. Respiratory ¹⁴CO₂ from the same animals is maximal at 3 hr, after which it falls off markedly. Approximately 30% of the injected radioactivity was recovered as ¹⁴CO₂ within 24 hr. The excretion of radioactivity after lipoate was administered by stomach tube was similar to that after intraperitoneal injection. Localization of radioactivity in the body was greatest in liver, intestinal contents, and muscle in all cases. Ionexchange and paper chromatographies of 24-hr pooled urine revealed several watersoluble radioactive metabolites. Incubation of [¹⁴C]lipoate with homogenates or mitochondrial preparations in vitro resulted in the production of ¹⁴CO₂, which was decreased by incubation with unlabeled fatty acids and unaffected by the addition of carnitine or (+)-decanoylcarnitine. The rat, like certain bacteria, metabolizes lipoate via β-oxidation of the valeric acid side chain and by other metabolic reactions on the dithiolane ring, which render the molecule more water soluble.     

Biosynthesis of lipoic acid in the rat: Incorporation of 35S- and 14C-labeled precursors

After injection of various ³⁵S- and ¹⁴C-containing compounds, the incorporation of the label into the lipoic acid present in the liver of growing rats has been determined. The best precursor for sulfur atoms, after 24 h, is cysteine; methionine and cystamine are scarcely incorporated and thiosulfate not at all. Good precursors of the carbon moiety are acetate and octanoate, whereas the incorporation of butyrate and cysteine is very low. It is concluded that lipoic acid is biosynthesized in the rat liver, and that sulfur atoms probably originate from cysteine.     

Scavenging of the cofactor lipoate is essential for the survival of the malaria parasite Plasmodium falciparum

Lipoate is an essential cofactor for key enzymes of oxidative metabolism. Plasmodium falciparum possesses genes for lipoate biosynthesis and scavenging, but it is not known if these pathways are functional, nor what their relative contribution to the survival of intraerythrocytic parasites might be. We detected in parasite extracts four lipoylated proteins, one of which cross-reacted with antibodies against the E2 subunit of apicoplast-localized pyruvate dehydrogenase (PDH). Two highly divergent parasite lipoate ligase A homologues (LplA), LipL1 (previously identified as LplA) and LipL2, restored lipoate scavenging in lipoylation-deficient bacteria, indicating that Plasmodium has functional lipoate-scavenging enzymes. Accordingly, intraerythrocytic parasites scavenged radiolabelled lipoate and incorporated it into three proteins likely to be mitochondrial. Scavenged lipoate was not attached to the PDH E2 subunit, implying that lipoate scavenging drives mitochondrial lipoylation, while apicoplast lipoylation relies on biosynthesis. The lipoate analogue 8-bromo-octanoate inhibited LipL1 activity and arrested P. falciparum in vitro growth, decreasing the incorporation of radiolabelled lipoate into parasite proteins. Furthermore, growth inhibition was prevented by lipoate addition in the medium. These results are consistent with 8-bromo-octanoate specifically interfering with lipoate scavenging. Our study suggests that lipoate metabolic pathways are not redundant, and that lipoate scavenging is critical for Plasmodium intraerythrocytic survival.     

Plasmid Involvement in Acyclic Isoprenoid Metabolism by Pseudomonas putida

An organism identified as Pseudomonas putida was found to utilize citronellol or geraniol as the sole carbon and energy source. The ability to degrade these acyclic isoprenols was associated with pSRQ50, a 50-megadalton transmissible plasmid.     

Kinetic analysis of the role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The catalytic roles of the two reductively acetylatable lipoic acid residues on each lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli were investigated. Both lipoyl groups are reductively acetylated from pyruvate at the same apparent rate and both can transfer their acetyl groups to CoASH, part-reactions of the overall complex reaction. The complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, conditions that lead to the modification and inactivation of the S-acetyldihydrolipoic acid residues. Modification was found to proceed appreciably faster than the accompanying loss of enzymic activity. The kinetics of the modification were fitted best by supposing that the two lipoyl groups react with the maleimide at different rates, one being modified at approximately 3.5 times the rate of the other. The loss of complex activity took place at a rate approximately equal to that calculated for the modification of the more slowly reacting lipoic acid residue. The simplest interpretation of this result is that only this residue is essential in the overall catalytic mechanism, but an alternative explanation in which one lipoic acid residue can take over the function of another was not ruled out. The kinetics of inactivation could not be reconciled with an obligatory serial interaction between the two lipoic acid residues. Similar experiments with the fluorescent N-[p-(benzimidazol-2-yl)phenyl]maleimide supported these conclusions, although the modification was found to be less specific than with N-ethylmaleimide. The more rapidly modified lipoic acid residue may be involved in the system of intramolecular transacetylation reactions that couple active sites in the lipoate acetyltransferase component.     

Desulfonation of Linear Alkylbenzenesulfonate Surfactants and Related Compounds by Bacteria

Pseudomonas putida S-313 (= DSM 6884) grew in sulfate-free medium when the sole sulfur source supplied was one of several arylsulfonates involved in the synthesis, application, or biodegradation of linear alkyl-benzenesulfonate (LAS) surfactants. 2-(4-Sulfophenyl)butyric acid, 4-n-butyl-1-methyl-6-sulfotetralin, and 4-toluenesulfonic acid were each completely utilized during growth, as were the model LAS 1-(4-sulfophenyl) octane and the arylsulfonate dyestuff Orange II. The product in each case was the corresponding phenol, which was identified by gas chromatography-mass spectrometry or ¹H nuclear magnetic resonance. Stoichiometric conversion of 4-toluenesulfonic acid to 4-cresol was observed. The molar growth yields observed were 2.4 to 2.8 kg of protein per mol of S, which were comparable to the yield for sulfate. Commercial LAS disappeared from growth medium inoculated with strain S-313, but negligible growth occurred; digestion of cells in alkali led to recovery of the LAS mixture, which seemingly sorbed to the cells. However, mixed culture L6 was readily obtained from batch enrichment cultures containing commercial LAS as a sole sulfur source and an inoculum from domestic sewage. Culture L6 desulfonated components of the LAS surfactant to the corresponding phenols, which were identified by gas chromatography-mass spectrometry. Compounds with shorter alkyl chains were desulfonated preferentially, as were the centrally substituted isomers. In the presence of 200 μM sulfate, culture L6 grew well and LAS disappeared, although this was due purely to sorption, as shown by digestion of the cells in alkali. Thus, under sulfate-limited conditions, LAS can be desulfonated directly.     

A novel amidotransferase required for lipoic acid cofactor assembly in Bacillus subtilis

In the companion paper we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate : protein ligase respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g. pyruvate dehydrogenase). We show that this function is provided by LipL, which catalyses the amidotransfer (transamidation) of the octanoyl moiety from octanoyl‐GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester‐linked acyl‐enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably uses a Cys‐Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.