Search

Pantothenate kinase (CoaA) catalyzes the first step of the eoenzyme A (CoA) biosynthetic pathway and controls the intracellular concentrations of CoA through feedback inhibition in bacteria. An alternative enzyme found in archaea, pantoate kinase, is missing in the order Thermoplasmatales. The PTO0232 gene from Picrophilus torridus, a thermoacidophilic euryarchaeon, is shown to be a distant homologue of the prokaryotic type I CoaA. The cloned gene clearly complements the poor growth of the temperature-sensitive Escherichia coli CoaA mutant strain ts9, and the recombinant protein expressed in E. coli cells transfers phosphate to pantothenate at pH 5 and 55 [degrees]C. In contrast to E. coli CoaA, the P. torridus enzyme is refractory to feedback regulation by CoA, indicating that in P. torridus cells the CoA levels are not regulated by the CoaA step. These data suggest the existence of two subtypes within the class of prokaryotic type I CoaAs. doi: 10.1128/JB.01021-09

In Salmonella enterica, the CobT enzyme activates the lower ligand base during the assembly of the nucleotide loop of adenosylcobalamin (AdoCbl) and other cobamides. Previously, mutational analysis identified a class of alleles (class M) that failed to restore AdoCbl biosynthesis during intragenic complementation studies. To learn why class M cobT mutations were deleterious, we determined the nature of three class M cobT alleles and performed in vivo and in vitro functional analyses guided by available structural data on the wild-type CobT ([CobT.sup.WT]) enzyme. We analyzed the effects of the variants CobT(G257D), CobT(GI71D), CobT(G320D), and CobT(C160A). The latter was not a class M variant but was of interest because of the potential role of a disulfide bond between residues C160 and C256 in CobT activity. Substitutions G171D, G257D, and G320D had profound negative effects on the catalytic efficiency of the enzyme. The C160A substitution rendered the enzyme fivefold less efficient than [CobT.sup.WT]. The CobT(G320D) protein was unstable, and results of structure-guided site-directed mutagenesis suggest that either variants CobT(G257D) and CobT(G171D) have less affinity for 5,6-dimethylbenzimidazole (DMB) or access of DMB to the active site is restricted in these variant proteins. The reported lack of intragenic complementation among class M cobT alleles is caused in some cases by unstable proteins, and in others it may be caused by the formation of dimers between two mutant CobT proteins with residual activity that is so low that the resulting CobT dimer cannot synthesize sufficient product to keep up with even the lowest demand for AdoCbl. doi: 10.1128/JB.01159-09

Propionyl-CoA is an inhibitor of both primary and secondary metabolism in Aspergillus species and a functional methylcitrate cycle is essential for the efficient removal of this potentially toxic metabolite. Although the genomes of most sequenced fungal species appear to contain genes coding for enzymes of the methylcitrate cycle, experimental confirmation of pathway activity in filamentous fungi has only been provided for Aspergillus nidulans and Aspergillus fumigatus. In this study we demonstrate that pathogenic Fusarium species also possess a functional methylcitrate cycle. Fusarium solani appears highly adapted to saprophytic growth as it utilized propionate with high efficiency, whereas Fusarium verticillioides grew poorly on this carbon source. In order to elucidate the mechanisms of propionyl-CoA detoxification, we first identified the genes coding for methylcitrate synthase from both species. Despite sharing 96% amino acid sequence identity, analysis of the two purified enzymes demonstrated that their biochemical properties differed in several respects. Both methylcitrate synthases exhibited low [K.sub.m] values for propionyl-CoA, but that of F. verticillioides displayed significantly higher citrate synthase activity and greater thermal stability. Activity determinations from cell-free extracts of F. solani revealed a strong methylcitrate synthase activity during growth on propionate and to a lesser extent on Casamino acids, whereas activity by F. verticillioides was highest on Casamino acids. Further phenotypic analysis confirmed that these biochemical differences were reflected in the different growth behaviour of the two species on propionyl-CoA-generating carbon sources. DOI 10.1099/mic.0.031781-0

In Bacillus anthracis, the novel type III pantothenate kinase ([PanK.sub.Ba]; encoded by coaX) catalyzes the first committed step in coenzyme A biosynthesis. We have demonstrated by analyzing the growth characteristics of a conditional coaX mutant that [PanK.sub.Ba] is an essential enzyme, thus contributing to its validation as a new antimicrobial target.

Amyotrophic lateral sclerosis (ALS), one of the most common adult-onset neurodegenerative diseases, has no known cure. Enhanced redox stress and inflammation have been associated with the pathoprogression of ALS through [...]

Mycobacteria can tolerate relatively high concentrations of triphenylmethane dyes such as malachite green and methyl violet. To identify mycobacterial genes involved in the decolorization of malachite green, a transposon mutant library of Mycobacterium smegmatis [mc.sup.2] 155 was screened for mutants unable to decolorize this dye. One of the genes identified was MSMEG_5126, an orthologue of Mycobacterium bovis fbiC encoding a 7,8-didemethyl-8hydroxy-5-deazariboflavin (FO) synthase, which is essential for the biosynthesis of the electron carrier coenzyme [F.sub.420]. The other gene identified was MSMEG_2392, encoding an alanine-rich protein with a DUF121 domain. The minimum inhibitory concentrations (MICs) for malachite green and methyl violet of the six fbiC mutants and two MSMEG_2392 mutants were one-third and one-fifth, respectively, of the MIC of the parent strain M. smegmatis [mc.sup.2] 155. Representative fbiC and MSMEG_2392 mutant strains were also sensitive to oxidative stress caused by the redox-cycling agents plumbagin and menadione, and the sensitivity was reversed in the complemented strains. HPLC analysis of representative fbiC and MSMEG_2392 strains revealed that, while the fbiC mutant lacked both coenzyme [F.sub.420] and FO, the MSMEG_2392 mutant contained FO but not coenzyme [F.sub.420]. These results indicate that MSMEG_2392 is involved in the biosynthesis of coenzyme [F.sub.420].

The use of molecular hydrogen as electron donor for energy generation is a defining characteristic of the hydrogenotrophic methanogens, an ancient group that dominates the phylum Euryarchaeota. We present here a global study of changes in mRNA abundance in response to hydrogen availability for a hydrogenotrophic methanogen. Cells of Methanococcus maripaludis were grown by using continuous culture to deconvolute the effects of hydrogen limitation and growth rate, and microarray analyses were conducted. Hydrogen limitation markedly increased mRNA levels for genes encoding enzymes of the methanogenic pathway that reduce or oxidize the electron-carrying deazaflavin, coenzyme [F.sub.420]. [F.sub.420]-dependent redox functions in energy-generating metabolism are characteristic of the methanogenic Archaea, and the results show that their regulation is distinct from other redox processes in the cell. Rapid growth increased mRNA levels of the gene for an unusual hydrogenase, the hydrogen-dependent methylenetetrahydromethanopterin dehydrogenase. coenzyme [F.sub.420] | microarrays | Methanococcus maripaludis

We report that cobC strains of Salmonella enterica serovar Typhimurium are impaired in the ability to salvage cobyric acid (Cby), a de novo coffin ring biosynthetic intermediate, under aerobic growth conditions. In vivo and in vitro evidence support the conclusion that this new phenotype of cobC strains is due to the inability of serovar Typhimurium to dephosphorylate adenosyleobalamin-5'-phosphate (AdoCbl-5'-P), the product of the condensation of [alpha]-ribazole-5'-phosphate [alpha]-RP) and adenosylcobinamide-GDP by the AdoCbl-5'-P synthase (CobS, EC 2.7.8.26) enzyme. Increased flux through the 5,6-dimethyibenzimidazole and cobinamide (Cbi) activation branches of the nucleotide loop assembly pathway in cobC strains restored AdoCbl-5'-P synthesis from Cby in a cobC strain. The rate of the CobS-catalyzed reaction was at least 2 orders of magnitude higher with [alpha]-RP than with [alpha]-ribazole as substrate. On the basis of the data reported herein, we conclude that removal of the phosphoryl group from AdoCbl-5'-P is the last step in AdoCbl biosynthesis in serovar Typhimurium and that the reaction is catalyzed by the AdoCbl-5'-P phosphatase (CobC) enzyme. Explanations for the correction of the Cby salvaging phenotype are discussed.

Coenzyme [Q.sub.10] (Co[Q.sub.10]) plays a pivotal role in oxidative phosphorylation (OXPHOS), as it distributes electrons among the various dehydrogenases and the cytochrome segments of the respiratory chain. We have identified [...]

The synthesis of 5,6-dimethylbenzimidazole (DMB), the lower ligand of coenzyme [B.sub.12], has remained elusive. We report in vitro and in vivo evidence that the BluB protein of the photosynthetic bacterium Rhodospirillum rubrum is necessary and sufficient for catalysis of the [O.sub.2]-dependent conversion of FMN[H.sub.2] to DMB. The product of the reaction (DMB) was isolated by using reverse-phase high-pressure liquid chromatography, and its identity was established by UV-visible spectroscopy and MS. No metals were detected in homogeneous preparations of BluB, and the enzyme did not affect DMB synthesis from 4,5-dimethylphenylenediamine and ribose-5-phosphate. The effect of the lack of bluB function in R. rubrum was reflected by the impaired ability of a [DELTA]bluB strain to convert Mg-protoporphyrin IX monomethyl ester (MPE) into protochlorophylide, a reaction of the bacteriochlorophyll biosynthetic pathway catalyzed by the MPE-cyclase enzyme present in this bacterium (BchE, EC 1.14.13.81), a predicted coenzyme [B.sub.12]- dependent enzyme. The growth defect of the [DELTA]bluB strain observed under anoxic photoheterotrophic conditions was corrected by the addition of DMB or [B.sub.12] to the culture medium or by introducing into the strain a plasmid encoding the wild-type allele of bluB. The findings reported here close an important gap in our understanding of the enzymology of the assembly of coenzyme [B.sub.12]. [B.sub.12] biosynthesis | [B.sub.12] lower ligand synthesis | oxygenases | vitamin metabolism

In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyciohexa-l,5-diene-l-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of [beta]-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophieus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA ([K.sub.m], 80 and 35 [micro]M; [V.sub.max], 350 and 550 [micro]mol [min.sup.-1] [mg.sup.-], respectively). Neither enzyme had significant activity with cyclohex-l-ene-l-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.

An infant with severe Leigh syndrome, nephrotic syndrome, and coenzyme [Q.sub.10] (Co[Q.sub.10]) deficiency in muscle and fibroblasts and compound heterozygous mutations in the decaprenyl diphosphate synthase subunit 2 (PDSS2) gene is described. Biochemical assays with radiolabeled substrates have indicated a severe defect in decaprenyl diphosphate synthase in the patient's fibroblasts.

Pantothenate kinase (PanK) catalyzes the first step in the five-step universal pathway of coenzyme A (CoA) biosynthesis, a key transformation that generally also regulates the intracellular concentration of CoA through feedback inhibition. A novel PanK protein encoded by the gene coaX was recently identified that is distinct from the previously characterized type I PanK (exemplified by the Escherichia coli coaA-encoded PanK protein) and type II eukaryotic PanKs and is not inhibited by CoA or its thioesters. This type III PanK, or PanK-III, is widely distributed in the bacterial kingdom and accounts for the only known PanK in many pathogenic species, such as Helicobacter pylori, Bordetella pertussis, and Pseudomonas aeruginosa. Here we report the first crystal structure of a type III PanK, the enzyme from Thermotoga maritima (Pan[K.sub.Tm]), solved at 2.0-[Angstrom], resolution. The structure of Pan[K.sub.Tm] reveals that type III PanKs belong to the acetate and sugar kinase/heat shock protein 70/actin (ASKHA) protein superfamily and that they retain the highly conserved active site motifs common to all members of this superfamily. Comparative structural analysis of the Pan[K.sub.Tm] active site configuration and mutagenesis of three highly conserved active site aspartates identify these residues as critical for PanK-III catalysis. Furthermore, the analysis also provides an explanation for the lack of CoA feedback inhibition by the enzyme. Since PanK-III adopts a different structural fold from that of the E. coli PanK--which is a member of the 'P-loop kinase' superfamily--this finding represents yet another example of convergent evolution of the same biological function from a different protein ancestor.

Abstract: Palm oil is known to host a variety of phytonutrients; some having antioxidant property such as the carotenes and vitamin E. These antioxidants are also present in the oil [...]

Flavin-dependent enzymes catalyse a wide range of reactions and, thereby, facilitate a variety of cellular processes. Among the properties that equip flavoenzymes with this chemical versatility is their reactivity towards oxygen, which shows huge variation among flavoproteins. A survey of known 3D structures of flavin-dependent oxidases and dehydrogenases and the correlation with their functional properties indicates that there are no structural rules that enable prediction of whether or how a flavoenzyme reacts with oxygen. Combinations of subtle factors such as dipole pre-organization, charge distribution, dynamics and solvation in the active centre determine the balance of interactions that control oxygen reactivity. The chemical basis of oxygen reactivity remains a puzzling problem and represents one of the challenging questions in modern flavoenzymology.

The 3-hydroxypropionate cycle has been proposed to operate as the autotrophic C[O.sub.2] fixation pathway in the phototrophic bacterium Chloroflexus aurantiucus. In this pathway, acetyl coenzyme A (acetyl-CoA) and two bicarbonate molecules are converted to malate. Acetyl-CoA is regenerated from malyl-CoA by L-malyl-CoA lyase. The enzyme forming malyl-CoA, succinyl-CoA:L-malate coenzyme A transferase, was purified. Based on the N-terminal amino acid sequence of its two subunits, the corresponding genes were identified on a gene cluster which also contains the gene for L-malyl-CoA lyase, the subsequent enzyme in the pathway. Both enzymes were severalfold up-regulated under autotrophic conditions, which is in line with their proposed function in C[O.sub.2] fixation. The two CoA transferase genes were cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Succinyl-CoA:L-malate CoA transferase forms a large ([alpha][beta]), complex consisting of 46- and 44-kDa subunits and catalyzes the reversible reaction succinyl-CoA + L-malate [right arrow] succinate + L-malyl-CoA. It is specific for succinyl-CoA as the CoA donor but accepts L-citramalate instead of L-malate as the CoA acceptor: the corresponding D-stereoisomers are not accepted. The enzyme is a member of the class III of the CoA transferase family. The demonstration of the missing CoA transferase closes the last gap in the proposed 3-hydroxypropionate cycle.

The enzymes that contain radicals and/or catalyze reactions with radical intermediates and are present in anaerobes are described. The families of coenzyme [B.sub.12]- and S-adenosylmethionine (SAM)-dependent radical enzymes are the exceptions as some of them occur in aerobes.

Coenzyme [Q.sub.10] supplementation on age-related changes in oxidative stress and function of heart mitochondria in rats fed a polyunsaturated fatty acid (PUFA)-rich diet was investigated. Two groups of rats were led for 24 months on a PUFA-rich diet, differing in supplementation or not with coenzyme [Q.sub.10]. Animals were killed at 6, 12, or 24 months. Fatty-acid profile, hydroperoxides, x-tocopherol, coenzyme Q, calalase and glutathione peroxidase activities, and cytochromes a+[a.sub.3], b, c+[c.sub.1] and cytochrome c oxidase activity were measured. Coenzyme [Q.sub.10]supplemented animals showed lower hydroperoxide levels: higher content and/or activity of xtocopherol, coenzyme Q, and catalase; and a slightly lower decrease in mitochondrial function. According to that, previously reported positive effects of coenzyme Q supplementation on the life span of rats fed a PUFA-rich diet might be a consequence, at least in part, of a lower oxidative stress level and perhaps, to a minor extent, of a smaller decrease in mitochondrial function.

The kinetics of deuterium incorporation into 5'-deoxyadenosine (5'dA) during the reaction was followed by rapid chemical quench, using HPLC and electro spray mass spectrometry to analyze the 5'-dA formed. Results imply that a large inverse equilibrium secondary isotope effect is associated with the formation of 5'-da from adenosylcobalamin.

The visualization, by confocal Raman microscopy, of the intracellular distribution of two enzymes important in the immune eosinophil peroxidase (EPO) in eosinophils is reported. The effect of photobleaching of the Raman signal at 413.1 nm excitation on the reconstructed Raman images is discussed.

In Salmonella enterica, the last step of the synthesis of adenosylcobamide is catalysed by the cobalamin synthase enzyme encoded by the cobs gene of this bacterium. Overexpression of the S. enterica cobS gene in Escherichia coil elicited the accumulation of the phage shock protein PspA, a protein whose expression has been linked to membrane stress. Resolution of inner and outer membranes of S. enterica by isopycnic density ultracentrifugation showed Cobs activity associated with the inner membrane, a result that was confirmed using antibodies against CobS. Computer analysis of the predicted amino acid sequence of CobS suggested it was an integral membrane protein. Results of experiments performed with strains carrying plasmids encoding CobS-alkaline phosphatase or CobS-[beta]-galactosidase protein fusions were consistent with the membrane localization of the CobS protein. Modifications to the predicted model were made based on data obtained from experiments using protein fusions. The function encoded by the cobS orthologue in the methanogenic archaeon Methanobacterium thermoautotrophicum strain [DELTA]H compensated for the lack of CobS during cobalamin synthesis in cobS strains of S. enterica. Cobalamin synthase activity was also detected in a membrane preparation of M. thermoautotrophicum. It was concluded that the assembly of the nucleotide loop of adenosylcobamides in archaea and bacteria is a membrane-associated process. Possible reasons for the association of adenosylcobamide biosynthetic enzymes with the cell membrane are discussed.

Research findings indicate that the development of several nicotinamide adenine dinucleotide (NAD) biosynthetic routes during evolution leading to NAD synthesis. Results point out that three distinct precursors are involved in the biosynthetic process and that the multiple metabolic routes in higher eukaryotes may be the result of differences in tissue distribution and/or intracellular compartmentalization of NAD metabolism.

Results reveal that the interconversion of the tryptophan synthase associated aldimine and alpha-aminoacrylate species leads to a proton release. The alpha-aminoacrylate species exhibiting absorption maximum at 350 nanometers is the Schiff base linkage.

Results suggest that the mechanism by which CoA increases the rate of electron transfer and redox reaction, involving pyruvate:ferredoxin oxidoreductase, is by forming a highly reducing adduct with the hydroxyethyl-thiamin pyrophosphate radical and/or possibly CoA itself becoming part of the electron-transfer pathway.

The characterization of the distance and relative orientation of the C5' methyl group of 5'-deoxyadenosine in vitamin B12 coenzyme-dependent ethanolamine deaminase is described.

Methyl-coenzyme M reductase (MCR) is a catalyst for the formation of methyl-coenzyme M (CH3SCH2CH2SO3) from methane. The inactive form of the active site, factor 430, are denoted MCR(silent) and MCR(ox1-Silent). The cryoreduction of these two forms by gamma-irradiation at 77k is described.

Research has been conducted on the glutathionylcobalamin functioning as an intermediate in the biosynthesis of the active B (sub)12 coenzymes adenosylcobalamin and methylcobalamin. The stability of this compound has been examined in comparison to other thionylcobalamins.

The steady-state synthesis of acetyl-CoA synthase at variable CO/CO2 pressures is described, in terms of kinetic mechanisms.

The FMN that is covalently attached to the beta subunit of heterotetrameric sarcosine oxidase from Corynebacterium sp. P-1 is essential for enzyme activity. The FMN ring is attached to N(3) of His173 and to an unidentified cysteine residue.

Research addresses the origin and evolution of coenzymes as exemplified by their synthesis and utilization in the RNA world. Data point out that two RNA sequences with coenzyme-synthesizing activities can mediate the synthesis of coenzyme A, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide from their precursors.

Research has been conducted on the malonate decarboxylase from Klebsiella pneumoniae that contains acyl carrier protein MdcC with CoA prosthetic group attached to it. The phosphoribosyl-dephospho-coenzyme A thansferase reaction of the prosthetic group biosynthesis has been investigated and the results are discussed.

Research has been conducted on the malonate decarboxylase from Klebsiella pneumoniae that catalyzes malonate cleavage to acetate and CO (sub)2. The biosynthesis of the smallest subunit of malonate decarboxylase, acyl carrier protein has been carried out.

There are two exchangeable hydrogens in the ethanolamine ammonia-lyase cofactor complex from Salmonella typhimurium. Both come from the C5' methylene of coenzyme B(sub)12.

UDP-galactopyranose mutase from Escherichia coli was studied to determine the role of flavin adenine dinucleotide in its catalysis.

A coenzyme B12 analogue with unusual properties is described.

The photochemical reactions of coenzyme PQQ with benzyl alcohol derivatives are examined.

The focus of this review article is the synthesis of coenzyme A (CoA) and unusual derivatives of CoA esters and analogues that can interact with CoA and its ester-utilizing enzymes. Data are also presented on the mechanistic aspects of CoA interaction with analogues as they developed from 1960 to 1980s.

Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene (T4HN) from malonyl coenzyme A by a fungal iterative type I polyketide synthase, PKSI, has been studied resulting in the first successful detection of cell-free activity of multi-aromatic-ring PKS from fungi. The Colletotrichum lagenarium PKS1 gene that encodes the synthase was overexpressed in Aspergillus oryzae. The in vitro result of the study clearly identified malonyl CoA, not acetyl CoA, as the starter and the extender units in the T4HN formation.

Results show that Escherichia coli growing under catabolite repression conditions induce acetyl-CoA synthetase gene thus enabling cells to assimilate acetate. Data further indicate that acetate utilization is mediated by cyclic AMP levels, decreasing oxygen partial pressure, and the carbon flux.

Research has been conducted on the dehydrogenase genes from Methanococcus jannaschii and Methanothermus fervidus. The cloning and overexpression of these genes and testing of the gene products for their catalytic ability have been carried out.

Research has been conducted on the dienoyl-coenzyme A thiolesters. The investigation of the stereochemical course of these thiolesters' reduction is discussed.

The benefits of choosing coenzymes as elements in considering the thermodynamics of systems of biochemical reactions in a living cell where the concentrations of coenzymes are in steady states are discussed. When the concentrations of coenzymes are constant, their conservation equations are eliminated.

Research has been conducted on the polyhydroxybutyrate (PHB) synthases. The conversion of the beta-hydroxybutyryl coenzyme A to PHB is described.

Research has been conducted on the wild-type 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase. The binding of [1,2-(super)13 C]acetyl-CoA to HMG-CoA synthase is described.

Research has been conducted on the Escherichia coli cells. Results indicate that these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway.

Research has been conducted on the phenylacetate-coenzyme A ligase that catalyzes aerobic degradation of phenylacetate in Azoarcus evansii. The purification and characterization of this enzyme has been carried out.