Ability of thermophilic lactic acid bacteria to produce aroma compounds from amino acids

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an alpha-keto acid such as alpha-ketoglutarate (alpha-KG) as the amino group acceptor, and produced alpha-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces alpha-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without alpha-KG addition. In the presence of alpha-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced alpha-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from alpha-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an alpha-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the alpha-keto acid conversion to acids in L. helveticus and S. thermophilus is an alpha-keto acid dehydrogenase that produces acyl coenzymes A.     

Characterization of a thiamin diphosphate-dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae

The product of the ARO10 gene from Saccharomyces cerevisiae was initially identified as a thiamine diphosphate-dependent phenylpyruvate decarboxylase with a broad substrate specificity. It was suggested that the enzyme could be responsible for the catabolism of aromatic and branched-chain amino acids, as well as methionine. In the present study, we report the overexpression of the ARO10 gene product in Escherichia coli and the first detailed in vitro characterization of this enzyme. The enzyme is shown to be an efficient aromatic 2-keto acid decarboxylase, consistent with it playing a major in vivo role in phenylalanine, tryptophan and possibly also tyrosine catabolism. However, its substrate spectrum suggests that it is unlikely to play any significant role in the catabolism of the branched-chain amino acids or of methionine. A homology model was used to identify residues likely to be involved in substrate specificity. Site-directed mutagenesis on those residues confirmed previous studies indicating that mutation of single residues is unlikely to produce the immediate conversion of an aromatic into an aliphatic 2-keto acid decarboxylase. In addition, the enzyme was compared with the phenylpyruvate decarboxylase from Azospirillum brasilense and the indolepyruvate decarboxylase from Enterobacter cloacae. We show that the properties of the two phenylpyruvate decarboxylases are similar in some respects yet quite different in others, and that the properties of both are distinct from those of the indolepyruvate decarboxylase. Finally, we demonstrate that it is unlikely that replacement of a glutamic acid by leucine leads to discrimination between phenylpyruvate and indolepyruvate, although, in this case, it did lead to unexpected allosteric activation.     

Fatty acid production from amino acids and alpha-keto acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and alpha-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of alpha-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and alpha-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from alpha-keto acids only. BL2 also converted alpha-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and alpha-keto acids and that carbon metabolism is important in regulating this event.     

Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis

Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.     

Structure and functioning mechanism of transketolase

Studies of thiamine diphosphate-dependent enzymes appear to have commenced in 1937, with the isolation of the coenzyme of yeast pyruvate decarboxylase, which was demonstrated to be a diphosphoric ester of thiamine. For quite a long time, these studies were largely focused on enzymes decarboxylating α-keto acids, such as pyruvate decarboxylase and pyruvate dehydrogenase complexes. Transketolase, discovered independently by Racker and Horecker in 1953 (and named by Racker) [1], did not receive much attention until 1992, when crystal X-ray structure analysis of the enzyme from Saccharomyces cerevisiae was performed [2]. These data, together with the results of site-directed mutagenesis, made it possible to understand in detail the mechanism of thiamine diphosphate-dependent catalysis. Some progress was also made in studies of the functional properties of transketolase. The last review on transketolase, which was fairly complete, appeared in 1998 [3]. Therefore, the publication of this paper should not seem premature.     

Effects of liver failure on the enzymes in the branched-chain amino acid catabolic pathway

Branched-chain alpha-keto acid dehydrogenase (BCKDH) complex catalyzes the committed step of the catabolism of branched-chain amino acids (BCAA). The liver cirrhosis chemically induced in rats raised the activity of hepatic BCKDH complex and decreased plasma BCAA and branched-chain alpha-keto acid concentrations, suggesting that the BCAA requirement is increased in liver cirrhosis. Since the effects of liver cirrhosis on the BCKDH complex in human liver are different from those in rat liver, further studies are needed to clarify the differences between rats and humans. In the valine catabolic pathway, crotonase and beta-hydroxyisobutyryl-CoA hydrolase are very important to regulate the toxic concentration of mitochondrial methacrylyl-CoA, which occurs in the middle part of valine pathway and highly reacts with free thiol compounds. Both enzyme activities in human and rat livers are very high compared to that of BCKDH complex. It has been found that both enzyme activities in human livers were significantly reduced by liver cirrhosis and hepatocellular carcinoma, suggesting a decrease in the capability to dispose methacrylyl-CoA. The findings described here suggest that alterations in hepatic enzyme activities in the BCAA catabolism are associated with liver failure.     

Characterization of Thi9, a novel thiamine (Vitamin B1) transporter from Schizosaccharomyces pombe

Thiamine is an essential component of the human diet and thiamine diphosphate-dependent enzymes play an important role in carbohydrate metabolism in all living cells. Although the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe can derive thiamine from biosynthesis, both are also able to take up thiamine from external sources, leading to the down-regulation of the enzymes involved in its formation. We have isolated the S. pombe thiamine transporter Thi9 by genetic complementation of mutants defective in thiamine biosynthesis and transport. Thi9 localizes to the S. pombe cell surface and works as a high-affinity proton/thiamine symporter. The uptake of thiamine was reduced in the presence of pyrithiamine, oxythiamine, amprolium, and the thiazole part of thiamine, indicating that these compounds are substrates of Thi9. In pyrithiamine-resistant mutants, a conserved glutamate residue close to the first of the 12 transmembrane domains is exchanged by a lysine and this causes aberrant localization of the protein. Thiamine uptake is significantly increased in thiamine-deficient medium and this is associated with an increase in thi9(+) mRNA and protein levels. Upon addition of thiamine, the thi9(+) mRNA becomes undetectable within minutes, whereas the Thi9 protein appears to be stable. The protein is distantly related to transporters for amino acids, gamma-aminobutyric acid and polyamines, and not to any of the known thiamine transporters. We also found that the pyridoxine transporter Bsu1 has a marked contribution to the thiamine uptake activity of S. pombe cells.     

Peptide inhibitors MAP the way towards fighting anthrax pathogenesis

BCAAs (branched-chain amino acids) are indispensable (essential) amino acids that are required for body protein synthesis. Indispensable amino acids cannot be synthesized by the body and must be acquired from the diet. The BCAA leucine provides hormone-like signals to tissues such as skeletal muscle, indicating overall nutrient sufficiency. BCAA metabolism provides an important transport system to move nitrogen throughout the body for the synthesis of dispensable (non-essential) amino acids, including the neurotransmitter glutamate in the central nervous system. BCAA metabolism is tightly regulated to maintain levels high enough to support these important functions, but at the same time excesses are prevented via stimulation of irreversible disposal pathways. It is well known from inborn errors of BCAA metabolism that dysregulation of the BCAA catabolic pathways that leads to excess BCAAs and their alpha-keto acid metabolites results in neural dysfunction. In this issue of Biochemical Journal, Joshi and colleagues have disrupted the murine BDK (branched-chain alpha-keto acid dehydrogenase kinase) gene. This enzyme serves as the brake on BCAA catabolism. The impaired growth and neurological abnormalities observed in this animal show conclusively the importance of tight regulation of indispensable amino acid metabolism.     

Cloning and characterization of a novel beta-transaminase from Mesorhizobium sp. strain LUK: a new biocatalyst for the synthesis of enantiomerically pure beta-amino acids

A novel beta-transaminase gene was cloned from Mesorhizobium sp. strain LUK. By using N-terminal sequence and an internal protein sequence, a digoxigenin-labeled probe was made for nonradioactive hybridization, and a 2.5-kb gene fragment was obtained by colony hybridization of a cosmid library. Through Southern blotting and sequence analysis of the selected cosmid clone, the structural gene of the enzyme (1,335 bp) was identified, which encodes a protein of 47,244 Da with a theoretical pI of 6.2. The deduced amino acid sequence of the beta-transaminase showed the highest sequence similarity with glutamate-1-semialdehyde aminomutase of transaminase subgroup II. The beta-transaminase showed higher activities toward d-beta-aminocarboxylic acids such as 3-aminobutyric acid, 3-amino-5-methylhexanoic acid, and 3-amino-3-phenylpropionic acid. The beta-transaminase has an unusually broad specificity for amino acceptors such as pyruvate and alpha-ketoglutarate/oxaloacetate. The enantioselectivity of the enzyme suggested that the recognition mode of beta-aminocarboxylic acids in the active site is reversed relative to that of alpha-amino acids. After comparison of its primary structure with transaminase subgroup II enzymes, it was proposed that R43 interacts with the carboxylate group of the beta-aminocarboxylic acids and the carboxylate group on the side chain of dicarboxylic alpha-keto acids such as alpha-ketoglutarate and oxaloacetate. R404 is another conserved residue, which interacts with the alpha-carboxylate group of the alpha-amino acids and alpha-keto acids. The beta-transaminase was used for the asymmetric synthesis of enantiomerically pure beta-aminocarboxylic acids. (3S)-Amino-3-phenylpropionic acid was produced from the ketocarboxylic acid ester substrate by coupled reaction with a lipase using 3-aminobutyric acid as amino donor.     

Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad.

Proteus, Providencia, and Morganella species produce deaminases that generate alpha-keto acids from amino acids. The alpha-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5 alpha for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.     

Branched-chain alpha-keto acid catabolism via the gene products of the bkd operon in Enterococcus faecalis: a new, secreted metabolite serving as a temporary redox sink

Recently the bkd gene cluster from Enterococcus faecalis was sequenced, and it was shown that the gene products constitute a pathway for the catabolism of branched-chain alpha-keto acids. We have now investigated the regulation and physiological role of this pathway. Primer extension analysis identified the presence of a single promoter upstream of the bkd gene cluster. Furthermore, a putative catabolite-responsive element was identified in the promoter region, indicative of catabolite repression. Consistent with this was the observation that expression of the bkd gene cluster is repressed in the presence of glucose, fructose, and lactose. It is proposed that the conversion of the branched-chain alpha-keto acids to the corresponding free acids results in the formation of ATP via substrate level phosphorylation. The utilization of the alpha-keto acids resulted in a marked increase of biomass, equivalent to a net production of 0.5 mol of ATP per mol of alpha-keto acid metabolized. The pathway was active under aerobic as well as anaerobic conditions. However, under anaerobic conditions the presence of a suitable electron acceptor to regenerate NAD(+) from the NADH produced by the branched-chain alpha-keto acid dehydrogenase complex was required for complete conversion of alpha-ketoisocaproate. Interestingly, during the conversion of the branched-chain alpha-keto acids an intermediate was always detected extracellularly. With alpha-ketoisocaproic acid as the substrate this intermediate was tentatively identified as 1, 1-dihydroxy-4-methyl-2-pentanone. This reduced form of alpha-ketoisocaproic acid was found to serve as a temporary redox sink.     

Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase

Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.     

Blood-brain barrier transport of the alpha-keto acid analogs of amino acids

A number of alpha-keto acid analogs of amino acids have been found to penetrate the blood-brain barrier (BBB). Pyruvate, alpha-ketobutyrate, alpha-ketoisocaproate, and alpha-keto-gamma-methiolbutyrate all cross the BBB by a carrier-mediated process and by simple diffusion. Under normal physiological conditions, diffusion accounts for roughly 15% or less of total transport. Aromatic alpha-keto acids, phenylpyruvate, and p-hydroxyphenylpyruvate do not penetrate the BBB, nor do they inhibit the transport of other alpha-keto acids. Evidence based primarily on inhibition studies indicates that the carrier-mediated transport of alpha-keto acids occurs via the same carrier demonstrated previously for propionate, acetoacetate, and beta-hydroxybutyrate transport, commonly referred to as the monocarboxylate carrier. As a group, the alpha-keto acid analogs of the amino acids have the highest affinity for the carrier, followed by propionate and beta-hydroxybutyrate. Starvation for 4 days induces transport of alpha-keto acids, but transport is suppressed in rats fed commercial laboratory rations and subjected to portacaval shunts. The mitochondrial pyruvate translocator inhibitor alpha-cyanocinnamate has no effect on the BBB transport of alpha-keto acids.     

Alpha-keto acids are novel siderophores in the genera Proteus, Providencia, and Morganella and are produced by amino acid deaminases.

Growth promotion and iron transport studies revealed that certain alpha-keto acids generated by amino acid deaminases, by enterobacteria of the Proteus-Providencia-Morganella group (of the tribe Proteeae), show significant siderophore activity. Their iron-binding properties were confirmed by the chrome azurol S assay and UV spectra. These compounds form ligand-to-metal charge transfer bands in the range of 400 to 500 nm. Additional absorption bands of the enolized ligands at 500 to 700 nm are responsible for color formation. Siderophore activity was most pronounced with alpha-keto acids possessing an aromatic or heteroaromatic side chain, like phenylpyruvic acid and indolylpyruvic acid, resulting from deamination of phenylalanine and tryptophan, respectively. In addition, alpha-keto acids possessing longer nonpolar side chains, like alpha-ketoisocaproic acid or alpha-ketoisovaleric acid and even alpha-ketoadipic acid, also showed siderophore activity which was absent or negligible with smaller alpha-keto acids or those possessing polar functional groups, like pyruvic acid, alpha-ketobutyric acid, or alpha-ketoglutaric acid. The fact that deaminase-negative enterobacteria, like Escherichia coli and Salmonella spp., could not utilize alpha-keto acids supports the view that specific iron-carboxylate transport systems have evolved in members of the tribe Proteeae and are designed to recognize ferric complexes of both alpha-hydroxy acids and alpha-keto acids, of which the latter can easily be generated by L-amino acid deaminases in an amino acid-rich medium. Exogenous siderophores, like ferric hydroxamates (ferrichromes) and ferric polycarboxylates (rhizoferrin and citrate), were also utilized by members of the tribe Proteeae.     

Branched chain amino acids induce apoptosis in neural cells without mitochondrial membrane depolarization or cytochrome c release: implications for neurological impairment associated with maple syrup urine disease

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a deficiency in branched chain alpha-keto acid dehydrogenase that can result in neurodegenerative sequelae in human infants. In the present study, increased concentrations of MSUD metabolites, in particular alpha-keto isocaproic acid, specifically induced apoptosis in glial and neuronal cells in culture. Apoptosis was associated with a reduction in cell respiration but without impairment of respiratory chain function, without early changes in mitochondrial membrane potential and without cytochrome c release into the cytosol. Significantly, alpha-keto isocaproic acid also triggered neuronal apoptosis in vivo after intracerebral injection into the developing rat brain. These findings suggest that MSUD neurodegeneration may result, at least in part, from an accumulation of branched chain amino acids and their alpha-keto acid derivatives that trigger apoptosis through a cytochrome c-independent pathway.     

Chloride-dependent inhibition of vesicular glutamate uptake by alpha-keto acids accumulated in maple syrup urine disease

Maple syrup urine disease is a metabolic disorder caused by mutations of the branched chain keto acid dehydrogenase complex, leading to accumulation of alpha-keto acids and their amino acid precursors in the brain. We now report that alpha-ketoisovaleric, alpha-keto-beta-methyl-n-valeric and alpha-ketoisocaproic acids accumulated in the disease inhibit glutamate uptake into rat brain synaptic vesicles. The alpha-keto acids did not affect the electrochemical proton gradient across the membrane, suggesting that they interact directly with the vesicular glutamate carrier. Chloride anions have a biphasic effect on glutamate uptake. Low concentrations activate the uptake (0.2 to 8 mM), while higher concentrations are inhibitory. The alpha-keto acids inhibited glutamate uptake by a new mechanism, involving a change in the chloride dependence for the activation of glutamate uptake. The activation of glutamate uptake by low chloride concentrations was shifted toward higher concentrations of the anion in the presence of alpha-keto acids. Inhibition by alpha-keto acids was abolished at high chloride concentrations (20 to 80 mM), indicating that alpha-keto acids specifically change the stimulatory effect of low chloride concentrations. High glutamate concentrations also reduced the inhibition by alpha-keto acids, an effect observed both in the absence and in the presence of low chloride concentrations. The results suggest that in addition to their possible pathophysiological role in maple syrup urine disease, alpha-keto acids are valuable tools to study the mechanism of vesicular transport of glutamate.     

Asparagine transaminase from rat liver.

Asparagine transaminase has been purified about 200-fold from rat liver. The enzyme has a broad specificity toward both amino acids and alpha-keto acids. Thus, amino acids substituted in the beta position such as asparagine, S-methylcysteine, phenylalanine, cysteine, serine, and aspartate are substrates. The enzyme is also active with alanine, methionine, homoserine, alpha-aminobutyrate, glutamine, and leucine. The enzyme has a high affinity for glyoxylate but the affinity falls off markedly through the series glyoxylate, pyruvate, alpha-ketoburyrate, alpha-Keto acids substituted in the beta or gamma position, such as alpha-ketosuccinamate, phenylpyruvate, p-hydroxyphenylpyruvate, alpha-keto-gamma-methiolburyrate, and alpha-keto-gamma-hydroxybutyrate, are substrates for the enzyme. Amino acids or alpha-keto acids possessing a branch point at the beta carbon are inactive. Kinetic analysis of the asparagine glyoxylate transamination reaction is consistent with a ping-pong mechanism.     

Some analogies between prebiotic thermal conversions of glycine and metabolic pathways

The reaction schemes suggested earlier for thermal transformation of glycine into amino acids and carboxylic acids are considered in detail. Close analogy with some wide-spread biochemical reactions of amino acids is observed. The pathway suggested has some common stages with the tricarboxylic acid cycle and other metabolic processes. The possible role of alpha-imino or alpha-keto acids as prebiological analogs of pyridoxal-phosphate-containing enzymes is discussed. The thermal transformations of glycine under primitive Earth conditions could be considered as evolutionary precursors of some present-day metabolic pathways.