Coenzyme A Thioesters Research Articles

Rapid Biocatalytic Synthesis of Aromatic Acid CoA Thioesters by Using Microbial Aromatic Acid CoA Ligases.

Chemically labile ester linkages can be introduced into lignin by incorporation of monolignol conjugates, which are synthesized in planta by acyltransferases that use a coenzyme A (CoA) thioester donor and a nucleophilic monolignol alcohol acceptor. The presence of these esters facilitates processing and aids in the valorization of renewable biomass feedstocks. However, the effectiveness of this strategy is potentially limited by the low steady-state levels of aromatic acid thioester donors in plants. As part of an effort to overcome this, aromatic acid CoA ligases involved in microbial aromatic degradation were identified and screened against a broad panel of substituted cinnamic and benzoic acids involved in plant lignification. Functional fingerprinting of this ligase library identified four robust, highly active enzymes capable of facile, rapid, and high-yield synthesis of aromatic acid CoA thioesters under mild aqueous reaction conditions mimicking in planta activity.

A Novel Coenzyme A Analogue in the Anaerobic, Sulfate-Reducing, Marine Bacterium Desulfobacula toluolica Tol2T.

Coenzyme A (CoA) thioesters are formed during anabolic and catabolic reactions in every organism. Degradation pathways of growth-supporting substrates in bacteria can be predicted by differential proteogenomic studies. Direct detection of proposed metabolites such as CoA thioesters by high-performance liquid chromatography coupled with high-resolution mass spectrometry can confirm the reaction sequence and demonstrate the activity of these degradation pathways. In the metabolomes of the anaerobic sulfate-reducing bacterium Desulfobacula toluolica Tol2T grown with different substrates various CoA thioesters, derived from amino acid, fatty acid or alcohol metabolism, have been detected. Additionally, the cell extracts of this bacterium revealed a number of CoA analogues with molecular masses increased by 1 dalton. By comparing the chromatographic and mass spectrometric properties of synthetic reference standards with those of compounds detected in cell extracts of D. toluolica Tol2T and by performing co-injection experiments, these analogues were identified as inosino-CoAs. These CoA thioesters contain inosine instead of adenosine as the nucleoside. To the best of our knowledge, this finding represents the first detection of naturally occurring inosino-CoA analogues.

Acyl Glucuronide and Coenzyme A Thioester Metabolites of Carboxylic Acid-Containing Drug Molecules: Layering Chemistry with Reactive Metabolism and Toxicology.

Glucuronidation and CoA (coenzyme A) conjugation are common pathways for the elimination of carboxylic acid-containing drug molecules. In some instances, these biotransformations have been associated with toxicity (such as idiosyncratic hepatic injury, renal impairment, hemolytic anemia, gastrointestinal inflammation, and bladder cancer) attributed to, in part, the propensity of acyl glucuronides and acyl CoA thioesters to covalently modify biological macromolecules such as proteins and DNA. It is to be noted that, while acyl glucuronidation and CoA conjugation are indeed implicated in adverse effects, there are many safe drugs in the market that are cleared by these reactive pathways. It is therefore important that new molecular entities with carboxylic acid groups are evaluated for toxicity in a manner that is not unreasonably risk-averse. In the absence of truly predictable methods, therefore, the general approach is to apply a set of end points to generate a weight-of-evidence evaluation. In practice, the focus is to identify structural liabilities and provide structure-activity recommendations early in the program, at a stage where an attempt to improve reactive metabolism does not deoptimize other critical drug-quality criteria. This review will present a high-level overview of the chemistry of glucuronidation and CoA conjugation and provide a discussion of the possible mechanisms of adverse effects that have been associated with these pathways, as well as how such potential hazards are addressed while delivering a new chemical entity for clinical evaluation.

Redefining the coenzyme A transferase superfamily with a large set of manually annotated proteins.

The coenzyme A (CoA) transferases are a superfamily of proteins central to the metabolism of acetyl-CoA and other CoA thioesters. They are diverse group, catalyzing over a 100 biochemical reactions and spanning all three domains of life. A deeply rooted idea, proposed two decades ago, is these enzymes fall into three families (I, II, and III). Here we find they fall into different families, which we achieve by analyzing all CoA transferases characterized to date. We manually annotated 94 CoA transferases with functional information (including rates of catalysis for 208 reactions) from 97 publications. This represents all enzymes we could find in the primary literature, and it is double the number annotated in four protein databases (BRENDA, KEGG, MetaCyc, UniProt). We found family I transferases are not closely related to each other in terms of sequence, structure, and reactions catalyzed. This family is not even monophyletic. These problems are solved by regrouping the three families into six, including one family with many non-CoA transferases. The problem (and solution) became apparent only by analyzing our large set of manually annotated proteins. It would have been missed if we had used the small number of proteins annotated in UniProt and other databases. Our work is important to understanding the biology of CoA transferases. It also warns investigators doing phylogenetic analyses of proteins to go beyond information in databases.

The Role of Peroxisomal CoA Degradation in the Regulation of Bile Acid Metabolism

Coenzyme A (CoA) is an essential metabolic cofactor that activates fatty acids and other organic acids as CoA thioesters for a multitude of metabolic pathways in different subcellular compartments. For example, CoA is utilized in the bile acid synthesis pathway and for shortening of very long chain fatty acids in the peroxisomes. In the liver, CoA levels are tightly regulated and dynamically adjusted in response to changes in nutritional status and energy demands. Balance between CoA synthesis and degradation is responsible for the regulation of CoA. Three intracellular CoA diphosphohydrolases, Nudt7, Nudt8, and Nudt19, catalyze the degradation of CoA by catalyzing the hydrolysis of its pyrophosphate bond. Nudt7 is the major CoA-degrading enzyme in the liver and resides in the peroxisomes. This enzyme displays a broad substrate specificity; it is active against free CoA, short- and medium-chain acyl-CoAs, including acetyl-CoA, and the acyl-CoA derivatives of bile acids.Nudt7 expression and activity are regulated by nutritional state, as they both decrease upon fasting. Overexpression of Nudt7 in the liver was recently found to decrease the levels of a variety of short-chain acyl-CoAs and choloyl-CoA, and to decrease both the rate of peroxisomal fatty acid oxidation and the concentration of bile acids in the liver. To gain further insight into the role of Nudt7 in vivo, we generated Nudt7-/- mice and analyzed their lipid metabolism in response to a dietary challenge with a high fat, high cholesterol (western) diet. Deletion of Nudt7 caused a significant increase in total hepatic CoA levels in Nudt7-/- males compared to control mice.Targeted and untargeted metabolomics analysis of whole liver extracts revealed an increase in glutaryl- and adipoyl-CoA in the Nudt7-/- males, associated with an accumulation of glutaryl-carnitine and other short-chain dicarboxylic acylcarnitines and with a decrease in long-chain acyl carnitines, suggesting an increase in the rate of fatty acid oxidation. Pathway enrichment analysis revealed changes in bile acid metabolism in both male and female Nudt7-/- mice compared to gender-matched controls.Specifically, deletion of Nudt7 led to an accumulation of bile acids in the liver, gallbladder, and small intestine, which also correlated with higher serum levels of Fgf15. This increase in bile acids occurred without changes in the expression of genes involved in bile acid synthesis or transport and without a correspondent increase in the concentration of bile acid precursors 7α-hydroxycholesterol and 7α-hydroxycholestenone. These results suggest that higher CoA levels in the peroxisomes may mediate an increase in flux through the bile acid biosynthetic pathway by activating the last, peroxisomal steps in the pathway. Overall, analysis of lipid metabolism following manipulation of Nudt7 expression in mouse liver indicates that this enzyme contributes to the regulation of peroxisomal fatty acids and bile acid metabolism.

Regulation of the CoA Biosynthetic Complex Assembly in Mammalian Cells.

Coenzyme A (CoA) is an essential cofactor present in all living cells. Under physiological conditions, CoA mainly functions to generate metabolically active CoA thioesters, which are indispensable for cellular metabolism, the regulation of gene expression, and the biosynthesis of neurotransmitters. When cells are exposed to oxidative or metabolic stress, CoA acts as an important cellular antioxidant that protects protein thiols from overoxidation, and this function is mediated by protein CoAlation. CoA and its derivatives are strictly maintained at levels controlled by nutrients, hormones, metabolites, and cellular stresses. Dysregulation of their biosynthesis and homeostasis has deleterious consequences and has been noted in a range of pathological conditions, including cancer, diabetes, Reye’s syndrome, cardiac hypertrophy, and neurodegeneration. The biochemistry of CoA biosynthesis, which involves five enzymatic steps, has been extensively studied. However, the existence of a CoA biosynthetic complex and the mode of its regulation in mammalian cells are unknown. In this study, we report the assembly of all five enzymes that drive CoA biosynthesis, in HEK293/Pank1β and A549 cells, using the in situ proximity ligation assay. Furthermore, we show that the association of CoA biosynthetic enzymes is strongly upregulated in response to serum starvation and oxidative stress, whereas insulin and growth factor signaling downregulate their assembly.

Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site

Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.

The Role of Coenzyme A Degradation in the Regulation of Peroxisomal Metabolism

Coenzyme A (CoA) is an essential metabolic cofactor that activates fatty acids and other organic acids as CoA thioesters for a multitude of metabolic pathways in different subcellular compartments. For example, CoA is utilized in the bile acid synthesis pathway and for shortening of very long chain fatty acids in the peroxisomes. The regulation of CoA levels is essential to maintain metabolic flexibility in response to changes in nutritional status and energy demands. Balance between CoA synthesis and degradation is responsible for the regulation of CoA. While CoA synthesis is controlled at the pantothenate kinase step, the intracellular degradation of CoA is controlled by Nudix hydrolases. Nudt7 is a hepatic, peroxisomal Nudix hydrolase that specifically hydrolyzes CoA species into 3’,5’‐ADP and (acyl)phosphopantetheine. Nudt7 displays a broad substrate specificity; it is active against free CoA, short‐ and medium‐chain acyl‐CoAs, including acetyl‐CoA, and the acyl‐CoA derivatives of bile acids. Nudt7 expression and activity are regulated by nutritional state, as they both decrease upon fasting. Overexpression of Nudt7 in the liver was recently found to decrease the levels of a variety of short‐chain acyl‐CoAs and choloyl‐CoA in the liver, and to decrease the rate of peroxisomal β‐oxidation. To gain further insight into the role of Nudt7 in vivo, Nudt7−/− mice were generated and their lipid metabolism assessed. Deletion of Nudt7 caused a small but significant increase in total hepatic CoA levels in the fasted state. In particular, higher concentrations of malonyl‐and acetyl‐CoA were observed. Furthermore, livers from Nudt7−/− males contained higher levels of short‐chain fatty acids and short‐chain acylcarnitines, which, combined, suggest an increase in fatty acid oxidation. In the fed state, female Nudt7−/− mice exhibited a higher concentration of selected bile acids in the intestine, while feeding of a high fat high/cholesterol diet resulted in higher bile acid levels in the liver. This increase in bile acids occurred without changes in the expression of genes involved in bile acid synthesis or transport and without a correspondent increase in the concentration of bile acid precursors 7α‐hydroxycholesterol and 7α‐hydroxycholestenone. These results suggest that higher CoA levels in the peroxisomes may mediate an increase in flux through the bile acid biosynthetic pathway by activating of the last, peroxisomal steps in the pathway.Overall, analysis of lipid metabolism following manipulation of Nudt7 expression in mouse liver indicates that this enzyme contributes to the regulation of peroxisomal fatty acids and bile acid metabolism.Support or Funding InformationThis work was supported by West Virginia University’s School of Medicine startup foundation funding and NIH grant GM119528.

Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria.

ABSTRACTMost mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene (ipdAB) and an acyl-CoA reductase gene (ipdC). In the presence of cholesterol, ΔipdC and ΔipdAB mutants of either M. tuberculosis or Rhodococcus jostii strain RHA1 accumulated previously undescribed metabolites: 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A ΔfadE32 mutant of Mycobacterium smegmatis accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by ΔipdF and ΔechA20 mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a ΔipdAB M. tuberculosis mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in M. tuberculosis.

Coenzyme A thioester formation of 11- and 15-oxo-eicosatetraenoic acid

Release of arachidonic acid (AA) by cytoplasmic phospholipase A2 (cPLA2), followed by metabolism through cyclooxygenase-2 (COX-2) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH), results in the formation of the eicosanoids 11-oxo- and 15-oxo-eicosatetraenoic acid (oxo-ETE). Both 11-oxo- and 15-oxo-ETE have been identified in human biospecimens but their function and further metabolism is poorly described. The oxo-ETEs contain an α,β-unsaturated ketone and a free carboxyclic acid, and thus may form Michael adducts with a nucleophile or a thioester with the free thiol of Coenzyme A (CoA). To examine the potential for eicosanoid-CoA formation, which has not previously been a metabolic route examined for this class of lipids, we applied a semi-targeted neutral loss scanning approach following arachidonic acid treatment in cell culture and detected inducible long-chain acyl-CoAs including a predominant AA-CoA peak. Interestingly, a series of AA-inducible acyl-CoAs at lower abundance but higher mass, likely corresponding to eicosanoid metabolites, was detected. Using a targeted LC–MS/MS approach we detected the formation of CoA thioesters of both 11-oxo- and 15-oxo-ETE and monitored the kinetics of their formation. Subsequently, we demonstrated that these acyl-CoA species undergo up to four double bond reductions. We confirmed the generation of 15-oxo-ETE-CoA in human platelets via LC-high resolution MS. Acyl-CoA thioesters of eicosanoids may provide a route to generate reducing equivalents, substrates for fatty acid oxidation, and substrates for acyl-transferases through cPLA2-dependent eicosanoid metabolism outside of the signaling contexts traditionally ascribed to eicosanoid metabolites.

Coenzyme A and its derivatives: renaissance of a textbook classic.

In 1945, Fritz Lipmann discovered a heat-stable cofactor required for many enzyme-catalysed acetylation reactions. He later determined the structure for this acetylation coenzyme, or coenzyme A (CoA), an achievement for which he was awarded the Nobel Prize in 1953. CoA is now firmly embedded in the literature, and in students' minds, as an acyl carrier in metabolic reactions. However, recent research has revealed diverse and important roles for CoA above and beyond intermediary metabolism. As well as participating in direct post-translational regulation of metabolic pathways by protein acetylation, CoA modulates the epigenome via acetylation of histones. The organization of CoA biosynthetic enzymes into multiprotein complexes with different partners also points to close linkages between the CoA pool and multiple signalling pathways. Dysregulation of CoA biosynthesis or CoA thioester homoeostasis is associated with various human pathologies and, although the biochemistry of CoA biosynthesis is highly conserved, there are significant sequence and structural differences between microbial and human biosynthetic enzymes. Therefore the CoA biosynthetic pathway is an attractive target for drug discovery. The purpose of the Coenzyme A and Its Derivatives in Cellular Metabolism and Disease Biochemical Society Focused Meeting was to bring together researchers from around the world to discuss the most recent advances on the influence of CoA, its biosynthetic enzymes and its thioesters in cellular metabolism and diseases and to discuss challenges and opportunities for the future.

Methods for measuring CoA and CoA derivatives in biological samples.

CoA (coenzyme A) is a ubiquitous and essential cofactor that acts as an acyl group carrier in biochemical reactions. Apart from participating in numerous metabolic pathways as substrates and intermediates, CoA and a number of its thioester derivatives, such as acetyl-CoA, can also directly regulate the activity of proteins by allosteric mechanisms and by affecting protein acetylation reactions. Cellular levels of CoA and CoA thioesters change under various physiological and pathological conditions. Defective CoA biosynthesis is implicated in NBIA (neurodegeneration with brain iron accumulation). However, the exact role of CoA in the pathogenesis of NBIA is not well understood. Accurate and reliable assays for measuring CoA species in biological samples are essential for studying the roles of CoA and CoA derivatives in health and disease. The present mini-review discusses methods that are commonly used to measure CoA species in biological samples.

Changes in acetyl CoA levels during the early embryonic development of Xenopus laevis.

Coenzyme A (CoA) is a ubiquitous and fundamental intracellular cofactor. CoA acts as a carrier of metabolically important carboxylic acids in the form of CoA thioesters and is an obligatory component of a multitude of catabolic and anabolic reactions. Acetyl CoA is a CoA thioester derived from catabolism of all major carbon fuels. This metabolite is at a metabolic crossroads, either being further metabolised as an energy source or used as a building block for biosynthesis of lipids and cholesterol. In addition, acetyl CoA serves as the acetyl donor in protein acetylation reactions, linking metabolism to protein post-translational modifications. Recent studies in yeast and cultured mammalian cells have suggested that the intracellular level of acetyl CoA may play a role in the regulation of cell growth, proliferation and apoptosis, by affecting protein acetylation reactions. Yet, how the levels of this metabolite change in vivo during the development of a vertebrate is not known. We measured levels of acetyl CoA, free CoA and total short chain CoA esters during the early embryonic development of Xenopus laevis using HPLC. Acetyl CoA and total short chain CoA esters start to increase around midblastula transition (MBT) and continue to increase through stages of gastrulation, neurulation and early organogenesis. Pre-MBT embryos contain more free CoA relative to acetyl CoA but there is a shift in the ratio of acetyl CoA to CoA after MBT, suggesting a metabolic transition that results in net accumulation of acetyl CoA. At the whole-embryo level, there is an apparent correlation between the levels of acetyl CoA and levels of acetylation of a number of proteins including histones H3 and H2B. This suggests the level of acetyl CoA may be a factor, which determines the degree of acetylation of these proteins, hence may play a role in the regulation of embryogenesis.

The crystal structures of the tri-functional Chloroflexus aurantiacus and bi-functional Rhodobacter sphaeroides malyl-CoA lyases and comparison with CitE-like superfamily enzymes and malate synthases

BackgroundMalyl-CoA lyase (MCL) is a promiscuous carbon-carbon bond lyase that catalyzes the reversible cleavage of structurally related Coenzyme A (CoA) thioesters. This enzyme plays a crucial, multifunctional role in the 3-hydroxypropionate bi-cycle for autotrophic CO2 fixation in Chloroflexus aurantiacus. A second, phylogenetically distinct MCL from Rhodobacter sphaeroides is involved in the ethylmalonyl-CoA pathway for acetate assimilation. Both MCLs belong to the large superfamily of CitE-like enzymes, which includes the name-giving β-subunit of citrate lyase (CitE), malyl-CoA thioesterases and other enzymes of unknown physiological function. The CitE-like enzyme superfamily also bears sequence and structural resemblance to the malate synthases. All of these different enzymes share highly conserved catalytic residues, although they catalyze distinctly different reactions: C-C bond formation and cleavage, thioester hydrolysis, or both (the malate synthases).ResultsHere we report the first crystal structures of MCLs from two different phylogenetic subgroups in apo- and substrate-bound forms. Both the C. aurantiacus and the R. sphaeroides MCL contain elaborations on the canonical β8/α8 TIM barrel fold and form hexameric assemblies. Upon ligand binding, changes in the C-terminal domains of the MCLs result in closing of the active site, with the C-terminal domain of one monomer forming a lid over and contributing side chains to the active site of the adjacent monomer. The distinctive features of the two MCL subgroups were compared to known structures of other CitE-like superfamily enzymes and to malate synthases, providing insight into the structural subtleties that underlie the functional versatility of these enzymes.ConclusionsAlthough the C. aurantiacus and the R. sphaeroides MCLs have divergent primary structures (~37% identical), their tertiary and quaternary structures are very similar. It can be assumed that the C-C bond formation catalyzed by the MCLs occurs as proposed for malate synthases. However, a comparison of the two MCL structures with known malate synthases raised the question why the MCLs are not also able to hydrolyze CoA thioester bonds. Our results suggest the previously proposed reaction mechanism for malate synthases may be incomplete or not entirely correct. Further studies involving site-directed mutagenesis based on these structures may be required to solve this puzzling question.

Crystal Structures of Acetobacter aceti Succinyl-Coenzyme A (CoA):Acetate CoA-Transferase Reveal Specificity Determinants and Illustrate the Mechanism Used by Class I CoA-Transferases

Coenzyme A (CoA)-transferases catalyze transthioesterification reactions involving acyl-CoA substrates, using an active-site carboxylate to form covalent acyl anhydride and CoA thioester adducts. Mechanistic studies of class I CoA-transferases suggested that acyl-CoA binding energy is used to accelerate rate-limiting acyl transfers by compressing the substrate thioester tightly against the catalytic glutamate [White, H., and Jencks, W. P. (1976) J. Biol. Chem. 251, 1688-1699]. The class I CoA-transferase succinyl-CoA:acetate CoA-transferase is an acetic acid resistance factor (AarC) with a role in a variant citric acid cycle in Acetobacter aceti. In an effort to identify residues involved in substrate recognition, X-ray crystal structures of a C-terminally His(6)-tagged form (AarCH6) were determined for several wild-type and mutant complexes, including freeze-trapped acetylglutamyl anhydride and glutamyl-CoA thioester adducts. The latter shows the acetate product bound to an auxiliary site that is required for efficient carboxylate substrate recognition. A mutant in which the catalytic glutamate was changed to an alanine crystallized in a closed complex containing dethiaacetyl-CoA, which adopts an unusual curled conformation. A model of the acetyl-CoA Michaelis complex demonstrates the compression anticipated four decades ago by Jencks and reveals that the nucleophilic glutamate is held at a near-ideal angle for attack as the thioester oxygen is forced into an oxyanion hole composed of Gly388 NH and CoA N2″. CoA is nearly immobile along its entire length during all stages of the enzyme reaction. Spatial and sequence conservation of key residues indicates that this mechanism is general among class I CoA-transferases.

Epoxy Coenzyme A Thioester Pathways for Degradation of Aromatic Compounds

Aromatic compounds (biogenic and anthropogenic) are abundant in the biosphere. Some of them are well-known environmental pollutants. Although the aromatic nucleus is relatively recalcitrant, microorganisms have developed various catabolic routes that enable complete biodegradation of aromatic compounds. The adopted degradation pathways depend on the availability of oxygen. Under oxic conditions, microorganisms utilize oxygen as a cosubstrate to activate and cleave the aromatic ring. In contrast, under anoxic conditions, the aromatic compounds are transformed to coenzyme A (CoA) thioesters followed by energy-consuming reduction of the ring. Eventually, the dearomatized ring is opened via a hydrolytic mechanism. Recently, novel catabolic pathways for the aerobic degradation of aromatic compounds were elucidated that differ significantly from the established catabolic routes. The new pathways were investigated in detail for the aerobic bacterial degradation of benzoate and phenylacetate. In both cases, the pathway is initiated by transforming the substrate to a CoA thioester and all the intermediates are bound by CoA. The subsequent reactions involve epoxidation of the aromatic ring followed by hydrolytic ring cleavage. Here we discuss the novel pathways, with a particular focus on their unique features and occurrence as well as ecological significance.

Anaerobic Metabolism of Indoleacetate

The anaerobic metabolism of indoleacetate (indole-3-acetic acid [IAA]) in the denitrifying betaproteobacterium Azoarcus evansii was studied. The strain oxidized IAA completely and grew with a generation time of 10 h. Enzyme activities that transformed IAA were present in the soluble cell fraction of IAA-grown cells but were 10-fold downregulated in cells grown on 2-aminobenzoate or benzoate. The transformation of IAA did not require molecular oxygen but required electron acceptors like NAD(+) or artificial dyes. The first products identified were the enol and keto forms of 2-oxo-IAA. Later, polar products were observed, which could not yet be identified. The first steps likely consist of the anaerobic hydroxylation of the N-heterocyclic pyrrole ring to the enol form of 2-oxo-IAA, which is catalyzed by a molybdenum cofactor-containing dehydrogenase. This step is probably followed by the hydrolytic ring opening of the keto form, which is catalyzed by a hydantoinase-like enzyme. A comparison of the proteome of IAA- and benzoate-grown cells identified IAA-induced proteins. Owing to the high similarity of A. evansii with strain EbN1, whose genome is known, we identified a cluster of 14 genes that code for IAA-induced proteins involved in the early steps of IAA metabolism. These genes include a molybdenum cofactor-dependent dehydrogenase of the xanthine oxidase/aldehyde dehydrogenase family, a hydantoinase, a coenzyme A (CoA) ligase, a CoA transferase, a coenzyme B(12)-dependent mutase, an acyl-CoA dehydrogenase, a fusion protein of an enoyl-CoA hydratase and a 3-hydroxyacyl-CoA dehydrogenase, a beta-ketothiolase, and a periplasmic substrate binding protein for ABC transport as well as a transcriptional regulator of the GntR family. Five predicted enzymes form or act on CoA thioesters, indicating that soon after the initial oxidation of IAA and possibly ring opening, CoA thioesters are formed, and the carbon skeleton is rearranged, followed by a CoA-dependent thiolytic release of another CoA thioester. We propose a scheme of an anaerobic IAA metabolic pathway that ultimately leads to 2-aminobenzoyl-CoA or benzoyl-CoA.

Structure and Mechanism of the Diiron Benzoyl-Coenzyme A Epoxidase BoxB

The coenzyme A (CoA)-dependent aerobic benzoate metabolic pathway uses an unprecedented chemical strategy to overcome the high aromatic resonance energy by forming the non-aromatic 2,3-epoxybenzoyl-CoA. The crucial dearomatizing reaction is catalyzed by three enzymes, BoxABC, where BoxA is an NADPH-dependent reductase, BoxB is a benzoyl-CoA 2,3-epoxidase, and BoxC is an epoxide ring hydrolase. We characterized the key enzyme BoxB from Azoarcus evansii by structural and Mössbauer spectroscopic methods as a new member of class I diiron enzymes. Several family members were structurally studied with respect to the diiron center architecture, but no structure of an intact diiron enzyme with its natural substrate has been reported. X-ray structures between 1.9 and 2.5 Å resolution were determined for BoxB in the diferric state and with bound substrate benzoyl-CoA in the reduced state. The substrate-bound reduced state is distinguished from the diferric state by increased iron-ligand distances and the absence of directly bridging groups between them. The position of benzoyl-CoA inside a 20 Å long channel and the position of the phenyl ring relative to the diiron center are accurately defined. The C2 and C3 atoms of the phenyl ring are closer to one of the irons. Therefore, one oxygen of activated O(2) must be ligated predominantly to this proximate iron to be in a geometrically suitable position to attack the phenyl ring. Consistent with the observed iron/phenyl geometry, BoxB stereoselectively should form the 2S,3R-epoxide. We postulate a reaction cycle that allows a charge delocalization because of the phenyl ring and the electron-withdrawing CoA thioester.

Stable Isotope Labeling by Essential Nutrients in Cell Culture for Preparation of Labeled Coenzyme A and Its Thioesters

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [13C315N1]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [13C315N1]-pantothenate after three passages of the murine cells in culture. Charcoal−dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

Structural and Enzymatic Analysis of Orf6, a Novel Dehydratase from a Deep‐Sea Polyunsaturated Fatty Acid Synthase

The polyketide synthase (PKS) genes responsible for eicosapentanoic acid (EPA) biosynthesis in the deep‐sea bacterium, Photobacterium profundum, have been cloned and sequenced, being a total of five polyunsaturated fatty acid (pfa) genes (pfaA, pfaB, pfaC, pfaD and Orf6) enough for EPA production. One of these enzymes is Orf6, a putative thioesterase (TE) domain believed to perform the last step in the fatty acid biosynthesis, catalyzing the hydrolysis and release of the final product from the carrier protein. The purpose of our research is to make a structure‐function analysis of Orf6 TE using its three dimensional structure determined by X‐ray crystallography to guide the re‐engineering of the enzyme. In parallel, we are also performing in vitro enzymatic assays with its likely substrates loaded onto coenzyme A (CoA). The Orf6 structure determined at 1.0 Å resolution reveals a novel hot‐dog fold containing a conserved amino acid, Asp17, in the active site. Results from activity assays indicate that Orf6 does not hydrolyze the CoA thioesters of long‐chain fatty acids. Additional efforts include in vitro assays using CoA thioesters of short‐chain carboxylic acids or acyl carrier protein (ACP) thioesters as substrates for Orf6. Structural and mechanistic knowledge of this seemingly novel enzyme will be important for the design of lipid‐based drugs or the development of industrial applications like biofuels.