scholarly journals Uptake of α-Ketoglutarate by Citrate Transporter CitP Drives Transamination in Lactococcus lactis

2012 ◽  
Vol 79 (4) ◽  
pp. 1095-1101 ◽  
Author(s):  
Agata M. Pudlik ◽  
Juke S. Lolkema
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Expression System ◽  
Ph Profile ◽  
Content Type ◽  
Citrate Transporter ◽  
Keto Acids ◽  
Branched Chain ◽  
Mode Of Transport

ABSTRACTTransamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells ofLactococcus lactisIL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence ofl-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone.

scholarly journals Rerouting Citrate Metabolism in Lactococcus lactis to Citrate-Driven Transamination

2012 ◽  
Vol 78 (18) ◽  
pp. 6665-6673 ◽  
Author(s):  
Agata M. Pudlik ◽  
Juke S. Lolkema
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Aromatic Amino Acids ◽  
High Concentration ◽  
Content Type ◽  
Citrate Transporter ◽  
Keto Acids ◽  
Citrate Fermentation ◽  
Branched Chain

ABSTRACTOxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm ofLactococcus lactisILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049–4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases ofL. lactisaccept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transaminationin vivowas further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.

scholarly journals CodY-Regulated Aminotransferases AraT and BcaT Play a Major Role in the Growth of Lactococcus lactis in Milk by Regulating the Intracellular Pool of Amino Acids

2003 ◽  
Vol 69 (6) ◽  
pp. 3061-3068 ◽  
Author(s):  
Emilie Chambellon ◽  
Mireille Yvon
Keyword(s):  
Amino Acids ◽  
Growth Inhibition ◽  
Lactococcus Lactis ◽  
Double Mutant ◽  
Wild Type Strain ◽  
Aromatic Amino Acids ◽  
Nitrogen Sources ◽  
Nutritional Factors ◽  
Keto Acids ◽  
Branched Chain

ABSTRACT Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.

scholarly journals The d-2-Hydroxyacid Dehydrogenase Incorrectly Annotated PanE Is the Sole Reduction System for Branched-Chain 2-Keto Acids in Lactococcus lactis

2008 ◽  
Vol 191 (3) ◽  
pp. 873-881 ◽  
Author(s):  
Emilie Chambellon ◽  
Liesbeth Rijnen ◽  
Frédérique Lorquet ◽  
Christophe Gitton ◽  
Johan E. T. van Hylckama Vlieg ◽  
...  
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Random Mutagenesis ◽  
Physiological Role ◽  
Branched Chain Amino Acids ◽  
New Family ◽  
Activity Sequence ◽  
Keto Acids ◽  
Cheese Flavor ◽  
Branched Chain

ABSTRACT Hydroxyacid dehydrogenases of lactic acid bacteria, which catalyze the stereospecific reduction of branched-chain 2-keto acids to 2-hydroxyacids, are of interest in a variety of fields, including cheese flavor formation via amino acid catabolism. In this study, we used both targeted and random mutagenesis to identify the genes responsible for the reduction of 2-keto acids derived from amino acids in Lactococcus lactis. The gene panE, whose inactivation suppressed hydroxyisocaproate dehydrogenase activity, was cloned and overexpressed in Escherichia coli, and the recombinant His-tagged fusion protein was purified and characterized. The gene annotated panE was the sole gene responsible for the reduction of the 2-keto acids derived from leucine, isoleucine, and valine, while ldh, encoding l-lactate dehydrogenase, was responsible for the reduction of the 2-keto acids derived from phenylalanine and methionine. The kinetic parameters of the His-tagged PanE showed the highest catalytic efficiencies with 2-ketoisocaproate, 2-ketomethylvalerate, 2-ketoisovalerate, and benzoylformate (V max/Km ratios of 6,640, 4,180, 3,300, and 2,050 U/mg/mM, respectively), with NADH as the exclusive coenzyme. For the reverse reaction, the enzyme accepted d-2-hydroxyacids but not l-2-hydroxyacids. Although PanE showed the highest degrees of identity to putative NADP-dependent 2-ketopantoate reductases (KPRs), it did not exhibit KPR activity. Sequence homology analysis revealed that, together with the d-mandelate dehydrogenase of Enterococcus faecium and probably other putative KPRs, PanE belongs to a new family of d-2-hydroxyacid dehydrogenases which is unrelated to the well-described d-2-hydroxyisocaproate dehydrogenase family. Its probable physiological role is to regenerate the NAD+ necessary to catabolize branched-chain amino acids, leading to the production of ATP and aroma compounds.

A General Method for Selection of α-Acetolactate Decarboxylase-Deficient Lactococcus lactis Mutants To Improve Diacetyl Formation

1999 ◽  
Vol 65 (3) ◽  
pp. 1202-1206 ◽  
Author(s):  
Mirjana Curic ◽  
Birgitte Stuer-Lauridsen ◽  
Pierre Renault ◽  
Dan Nilsson
Keyword(s):  
Amino Acids ◽  
Lactococcus Lactis ◽  
Genetically Modified Organisms ◽  
Defined Medium ◽  
Isoleucine Leucine ◽  
Genes Encoding ◽  
Resistant Mutants ◽  
Branched Chain ◽  
Selection Of

ABSTRACT The enzyme acetolactate decarboxylase (Ald) plays a key role in the regulation of the α-acetolactate pool in both pyruvate catabolism and the biosynthesis of the branched-chain amino acids, isoleucine, leucine, and valine (ILV). This dual role of Ald, due to allosteric activation by leucine, was used as a strategy for the isolation of Ald-deficient mutants of Lactococcus lactis subsp.lactis biovar diacetylactis. Such mutants can be selected as leucine-resistant mutants in ILV- or IV-prototrophic strains. Most dairy lactococcus strains are auxotrophic for the three amino acids. Therefore, the plasmid pMC004 containing the ilv genes (encoding the enzymes involved in the biosynthesis of IV) of L. lactis NCDO2118 was constructed. Introduction of pMC004 into ILV-auxotrophic dairy strains resulted in an isoleucine-prototrophic phenotype. By plating the strains on a chemically defined medium supplemented with leucine but not valine and isoleucine, spontaneous leucine-resistant mutants were obtained. These mutants were screened by Western blotting with Ald-specific antibodies for the presence of Ald. Selected mutants lacking Ald were subsequently cured of pMC004. Except for a defect in the expression of Ald, the resulting strain, MC010, was identical to the wild-type strain, as shown by Southern blotting and DNA fingerprinting. The mutation resulting in the lack of Ald in MC010 occurred spontaneously, and the strain does not contain foreign DNA; thus, it can be regarded as food grade. Nevertheless, its application in dairy products depends on the regulation of genetically modified organisms. These results establish a strategy to select spontaneous Ald-deficient mutants from transformable L. lactis strains.

scholarly journals Role of Aminotransferase IlvE in Production of Branched-Chain Fatty Acids by Lactococcus lactis subsp. lactis

2004 ◽  
Vol 70 (1) ◽  
pp. 638-641 ◽  
Author(s):  
Balasubramanian Ganesan ◽  
Bart C. Weimer
Keyword(s):  
Fatty Acids ◽  
Lactococcus Lactis ◽  
Deletion Mutant ◽  
Keto Acid ◽  
Branched Chain Amino Acid ◽  
Branched Chain Fatty Acids ◽  
Keto Acids ◽  
Branched Chain ◽  
Chain Fatty Acids

ABSTRACT The objective of this study was to determine the role of a lactococcal branched-chain amino acid aminotransferase gene, ilvE, in the production of branched-chain fatty acids. Lactococcus lactis subsp. lactis LM0230 and an ilvE deletion mutant, JLS450, produced branched-chain fatty acids from amino and α-keto acids at levels above α-keto acid spontaneous degradation and the fatty acids' flavor thresholds. The deletion mutant produced the same amounts of branched-chain fatty acids from precursor amino acids as did the parent. This was not the case, however, for the production of branched-chain fatty acids from the corresponding precursor α-keto acids. The deletion mutant produced a set of fatty acids different from that produced by the parent. We concluded from these observations that ilvE plays a role in the specific type of fatty acids produced but has little influence on the total amount of fatty acids produced by lactococci.

scholarly journals Plasma Branched-Chain Amino Acids and Incident Cardiovascular Disease in the PREDIMED Trial

2016 ◽  
Vol 62 (4) ◽  
pp. 582-592 ◽  
Author(s):  
Miguel Ruiz-Canela ◽  
Estefania Toledo ◽  
Clary B Clish ◽  
Adela Hruby ◽  
Liming Liang ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Amino Acids ◽  
Cardiovascular Death ◽  
Branched Chain Amino Acids ◽  
Control Group ◽  
Cvd Risk ◽  
Hazard Ratios ◽  
Increased Risk ◽  
Branched Chain

Abstract BACKGROUND The role of branched-chain amino acids (BCAAs) in cardiovascular disease (CVD) remains poorly understood. We hypothesized that baseline BCAA concentrations predict future risk of CVD and that a Mediterranean diet (MedDiet) intervention may counteract this effect. METHODS We developed a case-cohort study within the Prevención con Dieta Mediterránea (PREDIMED), with 226 incident CVD cases and 744 noncases. We used LC-MS/MS to measure plasma BCAAs (leucine, isoleucine, and valine), both at baseline and after 1 year of follow-up. The primary outcome was a composite of incident stroke, myocardial infarction, or cardiovascular death. RESULTS After adjustment for potential confounders, baseline leucine and isoleucine concentrations were associated with higher CVD risk: the hazard ratios (HRs) for the highest vs lowest quartile were 1.70 (95% CI, 1.05–2.76) and 2.09 (1.27–3.44), respectively. Stronger associations were found for stroke. For both CVD and stroke, we found higher HRs across successive quartiles of BCAAs in the control group than in the MedDiet groups. With stroke as the outcome, a significant interaction (P = 0.009) between baseline BCAA score and intervention with MedDiet was observed. No significant effect of the intervention on 1-year changes in BCAAs or any association between 1-year changes in BCAAs and CVD were observed. CONCLUSIONS Higher concentrations of baseline BCAAs were associated with increased risk of CVD, especially stroke, in a high cardiovascular risk population. A Mediterranean-style diet had a negligible effect on 1-year changes in BCAAs, but it may counteract the harmful effects of BCAAs on stroke.

scholarly journals ω-Amino Acid:Pyruvate Transaminase from Alcaligenes denitrificans Y2k-2: a New Catalyst for Kinetic Resolution of β-Amino Acids and Amines

2004 ◽  
Vol 70 (4) ◽  
pp. 2529-2534 ◽  
Author(s):  
Hyungdon Yun ◽  
Seongyop Lim ◽  
Byung-Kwan Cho ◽  
Byung-Gee Kim
Keyword(s):  
Amino Acids ◽  
Kinetic Resolution ◽  
Specific Activity ◽  
Expression System ◽  
Enantiomeric Excess ◽  
Selective Enrichment ◽  
Alcaligenes Denitrificans ◽  
Keto Acids ◽  
Optically Pure ◽  
High Stereoselectivity

ABSTRACT Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed ω-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic β-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for l-β-amino-n-butyric acid (l-β-ABA). The enzyme converts various β-amino acids and amines to the corresponding β-keto acids and ketones by using pyruvate as an amine acceptor. The apparent Km and V max for l-β-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM l-β-ABA, the apparent Km and V max for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM d,l-β-ABA, producing optically pure d-β-ABA (99% enantiomeric excess) with 53% conversion.

Role of insulin and branched-chain amino acids in regulating protein metabolism during fasting

1990 ◽  
Vol 258 (6) ◽  
pp. E907-E917 ◽  
Author(s):  
M. Frexes-Steed ◽  
M. L. Warner ◽  
N. Bulus ◽  
P. Flakoll ◽  
N. N. Abumrad
Keyword(s):  
Amino Acids ◽  
Protein Metabolism ◽  
Insulin Dose ◽  
Group Iii ◽  
Group I ◽  
Tissue Sensitivity ◽  
Branched Chain ◽  
Independent Effects ◽  
Dose Dependent

This study examines the independent effects of insulin and amino acids on protein metabolism after a 12-h and 4-day fast in healthy volunteers. Leucine (Leu) kinetics were examined during sequential insulin infusions of 0 (group I) or 0.0125 (groups II and III), 1.2, and 10 mU.kg-1.min-1. Plasma Leu was maintained at 12-h fasted levels in groups I and II and at 84-h fasted levels in group III. Four-day fast (vs. 1 day, P less than 0.01) was associated with a 79% drop in plasma insulin and elevations in plasma Leu (122%), Leu rates of appearance (Ra) (21%), and Leu oxidation (56%), and no change in nonoxidative rates of disappearance (Rd). Insulin resulted in a dose-dependent suppression of endogenous Leu Ra with group III = I greater than II. Leu oxidation rose 1.7-fold in group III at the highest insulin dose but remained stable in the two other groups. In conclusion, 4-day fasting is associated with enhanced proteolysis and Leu oxidation with no change in nonoxidative Rd (protein synthesis). Elevated branched-chain (and other) amino acids were required to restore tissue sensitivity and specificity to the effects of insulin on protein metabolism after 4 days of fasting.

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