Decarboxylation Of Α-keto Acids Research Articles

Recent Developments in Photoinduced Decarboxylative Acylation of α-Keto Acids.

Ketones are ubiquitous patterns found in various biological molecules and natural products. In recent years, a number of acylation methods have been developed based on the use of α-oxocarboxylic acids as acyl-transfer reagents, with particular emphasis on the photoinduced decarboxylative acylation of α-keto acids. This review focuses on the latest advancements in acylation methodologies through the decarboxylation of α-keto acids over the past several years, highlighting their product diversity, selectivity, and applicability. Where possible, the mechanistic rationale is presented, providing a positive outlook for the promising future of this field.

Novel cathodic and anodic dual-emitting electrochemiluminescence of Ru(bpy)32+/α-keto acid system.

Novel cathodic and anodic dual-emitting electrochemiluminescence (ECL) of Ru(bpy)32+ and α-keto acids system are studied for the first time. Based on their cathodic and anodic ECL intensity, α-keto acids including oxalate, glyoxylic acid, pyruvic acid, and phenylglyoxylic acid can be directly sensitively detected. The limits of detection (LOD) of oxalate, glyoxylic acid, pyruvic acid, and phenylglyoxylic acid are 31.25nM, 23.26µM, 36.36µM, and 18.52µM, respectively. Possible mechanism of ECL produced is also proposed. Electrochemical results show that the reduction of oxygen at the cathode to produce ·OH is a vital step for cathodic and anodic dual-emitting ECL. Furthermore, using the enhancement strategy of S2O82-/Ag+ as coreactant accelerators is proposed considering that decarboxylation of α-keto acids to produce acyl radical can be achieved via S2O82- or Ag+. Using the S2O82-/Ag+ enhancement strategy, the LOD of oxalate, glyoxylic acid, pyruvic acid, and phenylglyoxylic acid are improved and are 2.12nM, 0.37µM, 3.23µM, and 0.28µM, respectively. Coreactants of Ru(bpy)32+ with dual-emitting ECL are expanded, which includes additional substances with organic carboxylic acid characterized by the keto group in α-position. It also provides an effective way to enhance ECL and improve sensitivity. More importantly, cathodic and anodic dual-emitting ECL greatly improves the selectivity.

Structures and comparison of endogenous 2-oxoglutarate and pyruvate dehydrogenase complexes from bovine kidney

The α-keto acid dehydrogenase complex family catalyzes the essential oxidative decarboxylation of α-keto acids to yield acyl-CoA and NADH. Despite performing the same overarching reaction, members of the family have different component structures and structural organization between each other and across phylogenetic species. While native structures of α-keto acid dehydrogenase complexes from bacteria and fungi became available recently, the atomic structure and organization of their mammalian counterparts in native states remain unknown. Here, we report the cryo-electron microscopy structures of the endogenous cubic 2-oxoglutarate dehydrogenase complex (OGDC) and icosahedral pyruvate dehydrogenase complex (PDC) cores from bovine kidney determined at resolutions of 3.5 Å and 3.8 Å, respectively. The structures of multiple proteins were reconstructed from a single lysate sample, allowing direct structural comparison without the concerns of differences arising from sample preparation and structure determination. Although native and recombinant E2 core scaffold structures are similar, the native structures are decorated with their peripheral E1 and E3 subunits. Asymmetric sub-particle reconstructions support heterogeneity in the arrangements of these peripheral subunits. In addition, despite sharing a similar monomeric fold, OGDC and PDC E2 cores have distinct interdomain and intertrimer interactions, which suggests a means of modulating self-assembly to mitigate heterologous binding between mismatched E2 species. The lipoyl moiety lies near a mobile gatekeeper within the interdomain active site of OGDC E2 and PDC E2. Analysis of the twofold related intertrimer interface identified secondary structural differences and chemical interactions between icosahedral and cubic geometries of the core. Taken together, our study provides a direct structural comparison of OGDC and PDC from the same source and offers new insights into determinants of interdomain interactions and of architecture diversity among α-keto acid dehydrogenase complexes.

Direct Aroylation of Olefins through a Cobalt/Photoredox-Catalyzed Decarboxylative and Dehydrogenative Coupling with α-Oxo Acids.

A photoredox/cobalt dual catalytic procedure has been developed that allows benzoylation of olefins. Here the photoredox catalyst effects the decarboxylation of α-ketoacids to form benzoyl radicals. After addition of this radical to styrenes, the cobalt catalyst abstracts a H-atom. Hydrogen evolution from the putative cobalt hydride intermediate allows a Heck-like aroylation without the need for a stoichiometric oxidant. Mechanistic studies reveal that electronically different styrenes lead to a curved Hammett plot, thus suggesting a change in product-determining step in the catalytic mechanism.

Selective photoredox decarboxylation of α-ketoacids to allylic ketones and 1,4-dicarbonyl compounds dependent on cobaloxime catalysis.

A photoredox/cobaloxime co-catalyzed coupling reaction of α-ketoacids and methacrylates to obtain allylic ketones is described. Without the cobaloxime catalyst, 1,4-dicarbonyl compounds are generated. The cobaloxime catalyst enables dehydrogenation to generate the formation of new olefins. The generality, good substrate scope and mild conditions are good features in the photoredox/cobaloxime catalysis protocol, and this method will provide new opportunities for the functionalization of more olefins.

PSXIV-19 Effect of alpha-lipoic acid on productive parameters and ruminal VFA profile in sheep

Abstract Alpha lipoic acid (LA) is a potent nutritional antioxidant. It is a derivate of octanoic acid containing a cyclic disulfide bond. An LA complex with lysine-lipoamide, functions as a co-factor in the mitochondrial dehydrogenase that catalyzes the oxidative decarboxylation of α-keto acids. In this reaction, LA is reduced to dihydrolipoic acid (DHLA), which interacts with reactive oxygen species, and LA can also inactivate free radicals. Another important role of DHLA is in signal transduction by activating insulin receptors, which causes glucose transporters translocation from cytoplasm to the cell surface. Due to its potential antioxidant effect at ruminal level, the aim of this study was to evaluate the effect of fed LA on productive parameters, ruminal fatty volatile fatty acid (VFA) concentration and blood glucose of sheep. Sixty-four, two-months-old, crossbred lambs were penned in groups of four, divided into four treatments: 0, 40, 80, and 120 ppm LA in diet. During 84 days, every 28 days on two consecutive days, animals were weighed (to determine average daily weight gain, DWG), blood and ruminal fluid were taken for glucose and VFA quantification, respectively. All variables were analyzed in a completely randomized design. DWG was statistically higher in those animals receiving 80 ppm (250 g) as compared to the rest (200, 188, 180 g for 120, 40, 0 ppm, respectively) (P < 0.0001). No differences in blood glucose or ruminal VFA were found (P > 0.1). LA could improve the productive performance of sheep; however, it would be necessary to study the role of LA and its effects on the ruminal microbiome and its action mechanism. Research support by Project PAPIIT-UNAM IN211518

Structure-Guided Engineering of α-Keto Acid Decarboxylase for the Production of Higher Alcohols at Elevated Temperature.

Branched-chain keto acid decarboxylases (KDCs) are a class of enzymes that catalyze the decarboxylation of α-keto acids. They are key enzymes for production of higher alcohols in vivo and in vitro. However, the two most active KDCs (KivD and KdcA) have only moderate thermostability (<55 °C), which hinders the production of alcohols at high temperatures. Herein, structure-guided engineering toward improved thermostability of KdcA is outlined. Strategies such as stabilization of the catalytic center, surface engineering, and optimization of dimer interactions were applied. With seven amino acid substitutions, variant 7M.D showed an increase of the temperature at which 50 % of activity remains after one-hour incubation by 14.8 °C without compromising its substrate specificity. 7M.D exhibited greater than 400-fold improvement of half-life at 70 °C and greater than 600-fold increase in process stability in the presence of 4 % isobutanol at 50 °C. 7M.D is more promising for the production of higher alcohols in thermophiles (>65 °C) and in cell-free applications.

Ultrasound-enhanced Ag-catalyzed decarboxylative coupling between α-keto acids and disulfides for the synthesis of thioesters

Herein, we described the ultrasound-assisted synthesis of thioesters via the Ag-catalyzed radical oxidative decarboxylation of α-keto acids, in the presence of disulfides. This protocol takes advantage of the sonication to prepare the title compounds in moderate to very good yields, in only 20 min of reaction. The positive effect of ultrasonic irradiation is attributed to both, the high mass transfer efficiency and to the induced radical formation in the reaction medium.

Catalyst-free direct decarboxylative coupling of α-keto acids with thiols: a facile access to thioesters.

A novel, efficient, and catalyst-free strategy has been initially developed for the construction of thioesters via the direct radical oxidative decarboxylation of α-keto acids with thiols, and the corresponding target products were obtained in moderate to good yields. It offers an alternative approach for the synthesis of useful diverse thioesters.

A dual conformation of the post-decarboxylation intermediate is associated with distinct enzyme states in mycobacterial KGD (α-ketoglutarate decarboxylase)

α-Ketoacid dehydrogenases are large multi-enzyme machineries that orchestrate the oxidative decarboxylation of α-ketoacids with the concomitant production of acyl-CoA and NADH. The first reaction, catalysed by α-ketoacid decarboxylases (E1 enzymes), needs a thiamine diphosphate cofactor and represents the overall rate-limiting step. Although the catalytic cycles of E1 from the pyruvate dehydrogenase (E1p) and branched-chain α-ketoacid dehydrogenase (E1b) complexes have been elucidated, little structural information is available on E1o, the first component of the α-ketoglutarate dehydrogenase complex, despite the central role of this complex at the branching point between the TCA (tricarboxylic acid) cycle and glutamate metabolism. In the present study, we provide structural evidence that MsKGD, the E1o (α-ketoglutarate decarboxylase) from Mycobacterium smegmatis, shows two conformations of the post-decarboxylation intermediate, each one associated with a distinct enzyme state. We also provide an overall picture of the catalytic cycle, reconstructed by either crystallographic snapshots or modelling. The results of the present study show that the conformational change leading the enzyme from the initial (early) to the late state, although not required for decarboxylation, plays an essential role in catalysis and possibly in the regulation of mycobacterial E1o.

In Vivo Anti-inflammatory Activity of Lipoic Acid Derivatives in Mice

In mammals lipoic acid (LA) and its reduced form dihydrolipoic acid (DHLA) function as cofactors for multienzymatic complexes catalyzing the decarboxylation of α-ketoacids. Moreover, LA is used as a drug in a variety of diseases including inflammatory diseases. The aim of the study was to examine anti-inflammatory properties of LA metabolites. The present paper reports the chemical synthesis of 2,4-bismethylthio-butanoic acid (BMTBA) and tetranor-dihydrolipoic acid (tetranor-DHLA). BMTBA is one of the biotransformation products of LA, while tetranor-DHLA is an analogue of DHLA. Structural identity of these compounds was confirmed by 1H NMR. These compounds were assessed for their anti-inflammatory activity in mice. For this purpose, the zymosan-induced peritonitis and the carrageenan-induced hind paw edema animal models were applied. The obtained results indicated that the early vascular permeability measured at 30 min of zymosan-induced peritonitis was significantly inhibited in groups receiving BMTBA (10, 30, 50 mg/kg). The early infiltration of neutrophils measured at 4 hours of zymosan-induced peritonitis was inhibited in the group receiving BMTBA (50 mg/kg) and tetranor-DHLA (50 mg/kg). The results indicated that the increase in paw edema was significantly inhibited in the groups receiving BMTBA (50, 100 mg/kg) and tetranor-DHLA (30, 50 mg/kg). In summary, the present studies clearly demonstrated that both BMTBA and tetranor-DHLA were able to act as anti-inflammatory agents. This is the first study examining in vivo the anti-inflammatory properties of LA metabolites.

Ohr (Organic Hydroperoxide Resistance Protein) Possesses a Previously Undescribed Activity, Lipoyl-dependent Peroxidase

The Ohr (organic hydroperoxide resistance) family of 15-kDa Cys-based, thiol-dependent peroxidases is central to the bacterial response to stress induced by organic hydroperoxides but not by hydrogen peroxide. Ohr has a unique three-dimensional structure and requires dithiols, but not monothiols, to support its activity. However, the physiological reducing system of Ohr has not yet been identified. Here we show that lipoylated enzymes present in the bacterial extracts of Xylella fastidiosa interacted physically and functionally with this Cys-based peroxidase, whereas thioredoxin and glutathione systems failed to support Ohr peroxidase activity. Furthermore, we could reconstitute in vitro three lipoyl-dependent systems as the Ohr physiological reducing systems. We also showed that OsmC from Escherichia coli, an orthologue of Ohr from Xylella fastidiosa, is specifically reduced by lipoyl-dependent systems. These results represent the first description of a Cys-based peroxidase that is directly reduced by lipoylated enzymes.

Amino Acids Allosterically Regulate the Thiamine Diphosphate-dependent α-Keto Acid Decarboxylase from Mycobacterium tuberculosis

The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

Intermediates of thiamine catalysis immobilized on silica surface as active biocatalysts for α-ketoacid decarboxylation

Thiamine-dependent enzymes catalyse the decarboxylation of α-ketoacids, by both non-oxidative and oxidative mechanisms. Based on the ability of thiamine-cofactor to catalyse itself the decarboxylation of pyruvate to some extent, we have immobilized on a silica surface two ‘active aldehyde’ intermediates of thiamine catalysis, 2-α-hydroxybenzyl-thiamine pyrophosphate (HBTPP) and 2-α-hydroxyethyl-thiamine pyrophosphate (HETPP). The two intermediates have been tethered by a convenient method on silica support via their phosphate groups providing the covalently heterogenised biomolecules [HBTh–OP 2O 6–SiO 3/2] n · xSiO 2 and [HETh–OP 2O 6–SiO 3/2] n · xSiO 2. These bio-composite materials have been evaluated as catalysts for pyruvate and benzoyl-formate decarboxylation in either the presence or not of an aldehyde additive. Our data show that they are stable, very effective and recyclable catalysts for the production of 2-hydroxy-ketones, acetoine and benzoin. Their catalytic behavior is much better than the corresponding behavior of the homogeneous thiamine-systems due to the selected immobilization mode which bears similarities to that of the thiamine-cofactor binding to the protein. Considering our results, possible catalytic pathways of the prepared bio-composite materials are suggested.

Cell membrane damage induced by lacticin 3147 enhances aldehyde formation in Lactococcus lactis IFPL730

Amino acid catabolism is mainly initiated in Lactococcus lactis by a transamination reaction that leads to the formation of α-keto acids. In addition, a novel α-keto acid decarboxylase enzyme, rare in lactic acid bacteria, responsible for the conversion of α-keto acids into aldehydes has been reported in L. lactis IFPL730. The effect of lacticin 3147-induced cell damage on both amino acid transamination and α-keto acid decarboxylation by L. lactis IFPL730 leading to the formation of aldehydes from amino acids was investigated. Cell membrane permeabilization induced by lacticin 3147 facilitated the diffusion of amino acids into the cells and thus, enhanced amino acid transamination and formation of α-keto acids. However, α-keto acid decarboxylation was not affected by cell membrane permeabilization since decarboxylation of α-keto acids in both control and lacticin 3147-treated cells were similar, suggesting that these substrates could freely diffuse inside the cells. Nevertheless, the formation of 2-methylbutyraldehyde from isoleucine was enhanced in lacticin 3147-treated cells. The increase in α-keto acids formation rate by L. lactis IFPL730 due to lacticin 3147-induced cell damage, led to a concomitant increase in the subsequent decarboxylation reaction that complete the metabolic pathway to aldehyde production from amino acids. The present study points out to the use of the food grade lacticin 3147 along with L. lactis IFPL730 as a valuable tool in the development of cheese flavour.

A Versatile Conformational Switch Regulates Reactivity in Human Branched-Chain α-Ketoacid Dehydrogenase

The dehydrogenase/decarboxylase (E1b) component of the 4 MD human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is a thiamin diphosphate (ThDP)-dependent enzyme. We have determined the crystal structures of E1b with ThDP bound intermediates after decarboxylation of alpha-ketoacids. We show that a key tyrosine residue in the E1b active site functions as a conformational switch to reduce the reactivity of the ThDP cofactor through interactions with its thiazolium ring. The intermediates do not assume the often-postulated enamine state, but likely a carbanion state. The carbanion presumably facilitates the second E1b-catalyzed reaction, involving the transfer of an acyl moiety from the intermediate to a lipoic acid prosthetic group in the transacylase (E2b) component of the BCKDC. The tyrosine switch further remodels an E1b loop region to promote E1b binding to E2b. Our results illustrate the versatility of the tyrosine switch in coordinating the catalytic events in E1b by modulating the reactivity of reaction intermediates.

Modelling of l-DOPA enzymatic oxidation catalyzed by l-amino acid oxidases from Crotalus adamanteus and Rhodococcus opacus

l-amino acid oxidases ( l-AAO) are well known for their broad substrate specificity. l-amino acid oxidases from Crotalus adamanteus and Rhodococcus opacus were applied for biotransformation of 3,4-dihydroxyphenyl- l-alanine (L-DOPA) as a substrate to its corresponding α-keto acid. In this reaction, hydrogen peroxide formed as a by-product causes chemical decarboxylation of α-keto acids and acts as competitive product inhibitor. Beef liver catalase was used to decompose it. It was shown that both enzymes were able to oxidize l-DOPA to corresponding products. l-AAO from R. opacus was more specific (lower K m l − DOPA value) and more active towards l-DOPA substrate than l-AAO from C. adamanteus. Its catalytic constant, k 3, estimated by Levenspiel's method, was found to be 10-fold higher than the one for l-AAO from C. adamanteus. l-AAO from R. opacus exhibits slightly l-DOPA inhibition, which is not the case for l-AAO from C. adamanteus. The biotransformations of l-DOPA were carried out in batch enzyme membrane reactor (EMR), as well as in the repetitive batch EMR. The reactor and kinetics were modelled. Parameters were estimated by differential and integral method and presented in this article.

Effect of R(+)α-Lipoic acid on pyruvate metabolism and fatty acid oxidation in rat hepatocytes

R-(+)-α-lipoic acid (R-LA) is the naturally occuring enantiomer of LA. It is a strong antioxidant and cofactor of key metabolic enzyme complexes catalyzing the decarboxylation of α-keto acids. Racemic LA (rac-LA) has shown promise in treating diabetic polyneuropathy, and some studies suggest that it improves glucose homeostasis in patients with type 2 diabetes. We examined the effects of R-LA on pyruvate metabolism and free fatty acid (FFA) oxidation in primary cultured hepatocytes isolated from 24-hour fasted rats. After overnight culture in serum-free medium, cells were pre-exposed to R-LA for 3 hours before assays. R-LA (25 to 200 μmol/L) significantly increased pyruvate oxidation (∼2-fold at the highest dose tested) measured as 14CO 2 production from [1- 14C]pyruvate by the cells over 1 hour post-treatment. These effects correlated with proportional, significant increases in the activation state of the pyruvate dehydrogenase (PDH) complex. R-LA treatment inhibited glucose production from pyruvate by approximately 50% at 50 μmol/L R-LA and approximately 90% at 200 μmol/L. Palmitate oxidation was measured in hepatocytes cultured in the presence of albumin and physiological (0.1 mmol/L) or high (1.5 mmol/L) concentrations of FFA. The latter markedly enhanced FFA oxidation. R-LA treatment significantly inhibited FFA oxidation in both media, but was more effective in high FFA, where it reduced FFA oxidation by 48% to 82% at 25 to 200 μmol/L, respectively. Identical doses of R-LA did not affect FFA oxidation by L6 myotubes (a cell culture model for skeletal muscle) in either high or low FFA medium, but enhanced pyruvate oxidation. In conclusion, 3-hour exposure of primary cultured rat hepatocytes to R-LA at therapeutically relevant concentrations increased pyruvate oxidation, apparently by activation of the PDH complex, and decreased gluconeogenesis and FFA oxidation. These features may prove useful in the control of type 2 diabetes.

A Trail of Research from Lipoic Acid to α-Keto Acid Dehydrogenase Complexes

A Trail of Research from Lipoic Acid to α-Keto Acid Dehydrogenase Complexes

GROMOS-MD simulations on the coenzyme thiamin diphosphate in apoenzyme environment

Thiamin diphosphate (ThDP) is an essential cofactor for a number of enzymes, especially of pyruvate decarboxylase (PDC) which catalyzes the decarboxylation of α-keto acids. Recently, the crystal structure of PDC-bound ThDP has been determined. Based on these X-ray data molecular dynamics (MD) simulations of the isolated coenzyme as well as of ThDP in its enzymatic environment were performed, using the GROMOS87 software package. In the ThDP-apoenzyme model all significant amino acid residues with a cut-off radius less than 8.5 Å from the cofactor were considered. The conformational behavior and the formation of specific structures of both ThDP and enzyme-bound ThDP were investigated in order to get hints on the activity and the mechanism of the coenzyme. Therefore, trajectories of significant structural parameters were analyzed by our graphics tool. Moreover, Ramachandran-like plots with respect to significant torsion angles were used for the illustration. Finally, MD simulations on ThDP analogs with less or none catalytic activity and apoenzyme mutants were included, in order to study the cofactor-apoenzyme binding. © 1998 John Wiley & Sons, Inc. Int J Quant Chem 70: 407–413, 1998