Transient and long-term differential modulations of branched-chain α-keto acid decarboxylase activity in hypophysectomized rats

Hypophysectomy caused a marked but transient increase in branched-chain α-keto acid decarboxylase activities in rat liver mitochondria, peaking at about nine days post-surgery. The magnitude of increase is different for each of the three branched-chain α-keto acids. The activities then fall to a new steady state in three weeks with α-ketoisovalerate decarboxylase activity within the normal range, α-keto-β-methylvalerate decarboxylase activity at twice normal, and α-ketoisocaproate decarboxylase activity decreased to a level too low for accurate measurements.     

Biosynthesis of branched-chain fatty acids in Bacillus subtilis. A decarboxylase is essential for branched-chain fatty acid synthetase

Branched long-chain fatty acids of the iso and anteiso series are synthesized in many bacteria from the branched-chain alpha-keto acids of valine, leucine, and isoleucine after their decarboxylation followed by chain elongation. Two distinct branched-chain alpha-keto acid (BCKA) and pyruvate decarboxylases, which are considered to be responsible for primer synthesis, were detected in, and purified in homogenous form from Bacillus subtilis 168 strain by procedures including ammonium sulfate fractionation and chromatography on ion exchange, reversed-phase, and gel absorption columns. The chemical and catalytic properties of the two decarboxylases were studied in detail. The removal of BCKA decarboxylase, using chromatographic fractionation, from the fatty acid synthetase significantly reduced its activity. The synthetase activity was completely lost upon immunoprecipitation of the decarboxylase. The removal of pyruvate decarboxylase by the above two methods, however, did not affect any activity of the fatty acid synthetase. Thus, BCKA decarboxylase, but not pyruvate decarboxylase, is essential for the synthesis of branched-chain fatty acids. The very high affinity of BCKA decarboxylase toward branched-chain alpha-keto acids is responsible for its function in fatty acid synthesis.     

Growth hormone and liver mitochondria. Effects on cytochromes and some enzymes.

A reliable and reproducible assay was developed for measuring mitochondrial α-keto acid decarboxylase activity using ferricyanide as the electron acceptor. This method permitted the functional isolation and investigation of the decarboxylase step of the branched-chain α-keto acid dehydrogenases in rat liver mitochondria. Pyruvate and α-ketoglutarate decarboxylases are known to be separate and distinct enzymes from the branched-chain α-keto acid decarboxylases and were studied as controls. The relative specific activities of rat liver mitochondrial decarboxylases as measured by the ferricyanide assay showed that pyruvate and α-ketoglutarate were decarboxylated twice as rapidly as α-ketoisovalerate and four to ten times as fast as α-keto-β-methylvalerate and α-ketoisocaproate. The three branched-chain α-keto acids individually inhibit pyruvate and α-ketoglutarate decarboxylases. Inactivation of mitochondrial branched-chain α-keto acid decarboxylase activity by freezing and thawing and by prolonged storage resulted in a proportional decrease in decarboxylase activity toward each of the three branched-chain α-keto acids. However, hypophysectomy was found to increase decarboxylase activity with α-keto-β-methylvalerate to four times normal and with α-ketoisovalerate to three times normal, but the activity with α-ketoisocaproate was not changed. Hypophysectomy did not alter mitochondrial decarboxylase activity with pyruvate, α-ketoglutarate, or α-ketovalerate. The finding that hypophysectomy differentially alters the mitochondrial decarboxylase activity with the three branched-chain α-keto acids suggests either that there is more than one substrate-specific enzyme with branched-chain α-keto acid decarboxylase activity or that there is a modification of one enzyme such that the catalytic activity is selectively altered toward the three substrates.     

Identification, cloning, and characterization of a Lactococcus lactis branched-chain alpha-keto acid decarboxylase involved in flavor formation

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain alpha-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3' terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain alpha-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.     

A novel branched-chain amino acid metabolon. Protein-protein interactions in a supramolecular complex

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain alpha-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.     

Amino acids allosterically regulate the thiamine diphosphate-dependent alpha-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.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

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.     

Occurrence of a Tyr393----Asn (Y393N) mutation in the E1 alpha gene of the branched-chain alpha-keto acid dehydrogenase complex in maple syrup urine disease patients from a Mennonite population

Maple syrup urine disease (MSUD) is caused by a deficiency in the mitochondrial branched-chain alpha-keto acid dehydrogenase complex. The incidence of MSUD in the Philadelphia Mennonites is 1/176 births resulting from consanguinity. In this study, we amplified cDNAs for the decarboxylase E1 alpha subunit of the branched-chain alpha-keto acid dehydrogenase complex from a classical MSUD patient and from an obligatory heterozygote of a Mennonite family by the PCR. Sequencing of the amplified cDNAs disclosed at codon 393 of the mature E1 alpha polypeptide a base substitution changing a tyrosine (encoded by TAC) to an asparagine residue (encoded by AAC), which is designated Y393N. A segment of the E1 alpha gene containing the 5' portion of exon 9 was amplified. Probing of the amplified genomic DNA with allele-specific oligonucleotide probes showed that the mutation in the E1 alpha gene was homozygous in six Mennonites affected with classical MSUD and was present in heterozygous carriers. The identification of the MSUD mutation in the Philadelphia Mennonites will facilitate diagnosis and carrier detection for this population.     

Effect of starvation on branched-chain alpha-keto acid dehydrogenase activity in rat heart and skeletal muscle

The aim of the present study was to investigate changes in the activity of branched-chain alpha-keto acid dehydrogenase (BCKAD) in skeletal muscle and the heart during brief and prolonged starvation. Fed control rats and rats starved for 2, 4 and 6 days were anesthetized with pentobarbital sodium before heart and hindlimb muscles were frozen in situ by liquid nitrogen. Basal (an estimate of in vivo activity) and total (an estimate of enzyme amount) BCKAD activities were determined by measuring the release of 14CO2 from alpha-keto[1-(14)C]isocaproate. The activity state of BCKAD complex was calculated as basal activity in percentages of total activity. Both basal and total activities and the activity state of the BCKAD were lower in skeletal muscles than in the heart. In both tissues, starvation for 2 or 4 days caused a decrease in the basal activity and activity state of BCKAD. On the contrary, in the heart and muscles of animals starved for 6 days a marked increase in basal activity and activity state of BCKAD was observed. The total BCKAD activity was increasing gradually during starvation both in muscles and the heart. The increase was significant in muscles on the 4th and 6th day of starvation. The demonstrated changes in BCKAD activity indicate significant alterations in branched-chain amino acid (BCAA) and protein metabolism during starvation. The decreased BCKAD activity in skeletal muscle and heart observed on the 2nd and 4th day of starvation prevents the loss of essential BCAA and is an important factor involved in protein sparing. The increased activity of BCKAD on the 6th day of starvation indicates activated oxidation of BCAA and accelerated protein breakdown.     

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.     

Enzymatic determination of the branched-chain alpha-keto acids

A spectrophotometric endpoint assay for determination of branched-chain alpha-keto acids is described. The assay depends on measurement of the NADH produced after addition of branched-chain alpha-keto acid dehydrogenase. Interference by pyruvate and alpha-ketobutyrate was eliminated by pretreating the sample with pyruvate dehydrogenase. The method yielded a peripheral venous plasma value of 59 +/- 5 microM (mean +/- SE) for the branched-chain alpha-keto acids of five overnight fasted healthy humans.     

Biochemical and molecular characterization of alpha-ketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis

In this paper, we report for the first time on the identification, purification, and characterization of the alpha-ketoisovalerate decarboxylase from Lactococcus lactis, a novel enzyme responsible for the decarboxylation into aldehydes of alpha-keto acids derived from amino acid transamination. The kivd gene consisted of a 1647 bp open reading frame encoding a putative peptide of 61 kDa. Analysis of the deduced amino acid sequence indicated that the enzyme is a non-oxidative thiamin diphosphate (ThDP)-dependent alpha-keto acid decarboxylase included in the pyruvate decarboxylase group of enzymes. The active enzyme is a homo-tetramer that showed optimum activity at 45 degrees C and at pH 6.5 and exhibited an inhibition pattern typical for metal-dependant enzymes. In addition to Mg(2+), activity was observed in presence of other divalent cations such as Ca(2+), Co(2+) and Mn(2+). The enzyme showed the highest specific activity (80.7 Umg(-1)) for alpha-ketoisovalerate, an intermediate metabolite in valine and leucine biosynthesis. On the other side, decarboxylation of indole-3-pyruvate and pyruvate only could be detected by a 100-fold increase in the enzyme concentration present in the reaction.     

Isolation of a branched-chain alpha-keto acid decarboxylase from rat liver.

An enzyme which catalyses oxidative decarboxylation of branched-chain alpha-keto acids was extracted from rat liver mitochondria with the aid of NaClO4. Purification yielded a product which appeared homogenous upon electrophoresis. Some kinetic data are reported; however, the enzyme is inactive with alpha-ketoisovalerate. The tenacity of binding to mitochondria, specificity, and other features, suggest that the decarboxylase may be a component of an enzyme complex named alpha-ketoisocaproate: alpha-keto-beta-methylvalerate dehydrogenase.     

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.     

The effect of acylcarnitines on the oxidation of branched chain alpha-keto acids in mitochondria

The oxidation of 14C-labelled branched-chain alpha-keto acids corresponding to the branched-chain amino acids valine, isoleucine and leucine has been studied in isolated mitochondria from heart, liver and skeletal muscle. 1. Heart and liver mitochondria have similar capacities to oxidize these alpha-keto acids based on protein content. Skeletal muscle mitochondria also show significant activity. 2. Half maximum rates are obtained with approximately 0.1 mM of the alpha-keto acids under optimal conditions. Added NAD and CoA had no effect on the oxidation rate, showing that endogenous mitochondrial NAD and CoA are required for the oxidation. 3. Addition of carnitine esters of fatty acids (C6--C16), succinate, pyruvate, or alpha-ketoglutarate inhibited the oxidation of the branched chain alpha-keto acids, especially in a high-energy state (no ADP added). In heart mitochondria the addition of AD (low-energy state) decreased the inhibitory effects of acylcarnitines of medium chain length or of pyruvate, and abolished the inhibitory effect of succinate. It is suggested that the oxidation rate is regulated mainly by the redox state of the mitochondria under the conditions used. 4. The results are discussed in relation to the regulation of branched-chain amino acid metabolism in the body.     

Effects of dietary protein intake on branched-chain keto acid dehydrogenase activity of the rat. Immunochemical analysis of the enzyme complex

Polyclonal antibodies directed against the dihydrolipoyl transacylase (E2) and alpha subunit of branched-chain alpha-keto acid decarboxylase (E1 alpha) components of the bovine branched-chain keto acid dehydrogenase complex were shown to cross-react with the E2 and E1 alpha polypeptides of the enzyme complex of different rat tissues. Phosphorylation of the branched-chain keto acid dehydrogenase complex resulted in inhibition of enzyme activity concomitant with phosphate incorporation into the E1 alpha polypeptide. Phosphorylation of E1 alpha slowed its rate of migration through sodium dodecyl sulfate-polyacrylamide gels. This permitted resolution of the phosphorylated and unphosphorylated forms of E1 alpha on immunoblots. Liver and skeletal muscle mitochondria were prepared from rats consuming 6, 20, or 50% casein diets. The enzyme complex in mitochondria was measured by radioisotopic enzyme assay and immunoassay. Liver branched-chain keto acid dehydrogenase was 25% active in rats consuming 6% casein diets; whereas in rats consuming 20 or 50% casein diets, the liver enzyme was 82 or 100% active, respectively. Branched-chain keto acid dehydrogenase of muscle was 10, 13, and 22% active, respectively, in rats consuming 6, 20, and 50% casein diets. The amount of protein consumed by rats did not affect the total amount of the enzyme complex per unit of mitochondrial protein as measured by either the radioisotopic assay (enzyme activity) or the immunoassay. However, the protein intake of rats did affect activity of the enzyme kinase in liver. Liver branched-chain keto acid dehydrogenase kinase was more active in rats consuming 6% casein than in those fed chow or 50% casein diets. The amount of protein consumed by rats thus influences the enzyme activity in liver and muscle by affecting the reversible phosphorylation mechanism and not by induction of branched-chain keto acid dehydrogenase.     

The alpha-ketoisocaproate catabolism in human and rat livers

Catabolism of alpha-ketoisocaproate in liver is mediated by cytosolic alpha-ketoisocaproate dioxygenase (KICD) and mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). The latter is believed to be involved in the main pathway of the KIC catabolism. In the present study, we measured the activities of KICD and BCKDC in human and rat livers. The KICD activity in human liver was 0.9 mU/g tissue, which was 14.2% of the total activity of BCKDC, and that in rat liver was 4.2 mU/g tissue, which was only 1.0% of the total activity, suggesting that KICD in human liver plays a relatively important role in the alpha-ketoisocaproate catabolism. The KICD activity in human liver was significantly increased by cirrhosis. In rat liver, the enzyme activity was markedly increased by physical training and streptozotocin-induced diabetes, but not by feeding of a diet rich in branched-chain amino acids, although BCKDC activity was increased by feeding of the diet.     

Experimental hyperthyroidism causes inactivation of the branched-chain alpha-ketoacid dehydrogenase complex in rat liver

Hyperthyroidism induced by 3-day treatment of rats with thyroid hormone (T(3); 3,5,3'-triiodothyronine) at 0.1 or 1 mg/kg body wt/day resulted in a reduced activity state (% of enzyme in its active, dephosphorylated state) of the hepatic branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex. One treatment with 0.1 mg T(3)/kg body wt caused a significant effect on the activity state of BCKDH complex after 24 h, indicating that the reduction of the activity state was triggered by the first administration of T(3). Hyperthyroidism also caused a stable increase in BCKDH kinase activity, the enzyme responsible for phosphorylation and inactivation of the BCKDH complex, suggesting that T(3) caused inactivation of the BCKDH complex by induction of its kinase. Western blot analysis also revealed increased amounts of BCKDH kinase protein in response to hyperthyroidism. No change in the plasma levels of branched-chain alpha-keto acids was observed in T(3)-treated rats, arguing against an involvement of these known regulators of BCKDH kinase activity. Inactivation of the hepatic BCKDH complex as a consequence of overexpression of its kinase may save the essential branched-chain amino acids for protein synthesis during hyperthyroidism.     

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.