Functional Analysis of All Aminotransferase Proteins Inferred from the Genome Sequence of Corynebacterium glutamicum

ABSTRACT Twenty putative aminotransferase (AT) proteins of Corynebacterium glutamicum, or rather pyridoxal-5′-phosphate (PLP)-dependent enzymes, were isolated and assayed among others with l-glutamate, l-aspartate, and l-alanine as amino donors and a number of 2-oxo-acids as amino acceptors. One outstanding AT identified is AlaT, which has a broad amino donor specificity utilizing (in the order of preference) l-glutamate > 2-aminobutyrate > l-aspartate with pyruvate as acceptor. Another AT is AvtA, which utilizes l-alanine to aminate 2-oxo-isovalerate, the l-valine precursor, and 2-oxo-butyrate. A second AT active with the l-valine precursor and that of the other two branched-chain amino acids, too, is IlvE, and both enzyme activities overlap partially in vivo, as demonstrated by the analysis of deletion mutants. Also identified was AroT, the aromatic AT, and this and IlvE were shown to have comparable activities with phenylpyruvate, thus demonstrating the relevance of both ATs for l-phenylalanine synthesis. We also assessed the activity of two PLP-containing cysteine desulfurases, supplying a persulfide intermediate. One of them is SufS, which assists in the sulfur transfer pathway for the Fe-S cluster assembly. Together with the identification of further ATs and the additional analysis of deletion mutants, this results in an overview of the ATs within an organism that may not have been achieved thus far.

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Improved l-Leucine Production in Corynebacterium glutamicum by Optimizing the Aminotransferases

Molecules ◽

10.3390/molecules23092102 ◽

2018 ◽

Vol 23 (9) ◽

pp. 2102 ◽

Cited By ~ 6

Author(s):

Li-Yan Feng ◽

Jian-Zhong Xu ◽

Wei-Guo Zhang

Keyword(s):

Corynebacterium Glutamicum ◽

Branched Chain Amino Acids ◽

Gene Products ◽

Branched Chain Amino Acid ◽

Synthesis Activity ◽

Branched Chain ◽

Aromatic Aminotransferase ◽

First Time ◽

Branched Chain Aminotransferase

The production of branched-chain amino acids (BCAAs) is still challenging, therefore we rationally engineered Corynebacterium glutamicum FA-1 to increase the l-leucine production by optimizing the aminotransferases. Based on this, we investigated the effects of the native aminotransferases, i.e., branched-chain amino acid aminotransferase (BCAT; encoded by ilvE) and aspartate aminotransferase (AspB; encoded by aspB) on l-leucine production in C. glutamicum. The strain FA-1△ilvE still exhibited significant growth without leucine addition, while FA-1△ilvE△aspB couldn’t, which indicated that AspB also contributes to L-leucine synthesis in vivo and the yield of leucine reached 20.81 ± 0.02 g/L. It is the first time that AspB has been characterized for l-leucine synthesis activity. Subsequently, the aromatic aminotransferase TyrB and the putative aspartate aminotransferases, the aspC, yhdR, ywfG gene products, were cloned, expressed and characterized for leucine synthesis activity in FA-1△ilvE△aspB. Only TyrB was able to synthesize l-leucine and the l-leucine production was 18.55 ± 0.42 g/L. The two putative branched-chain aminotransferase genes, ybgE and CaIlvE, were also cloned and expressed. Both genes products function efficiently in BCAAs biosynthesis. This is the first report of a rational modification of aminotransferase activity that improves the l-leucine production through optimizing the aminotransferases.

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Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly

International Journal of Molecular Sciences ◽

10.3390/ijms22041598 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1598

Author(s):

Amber L. Hendricks ◽

Christine Wachnowsky ◽

Brian Fries ◽

Insiya Fidai ◽

James A. Cowan

Keyword(s):

Cluster Assembly ◽

Paramagnetic Resonance ◽

Iron Sulfur Cluster ◽

Sulfur Transfer ◽

Disease States ◽

Isolation And Characterization ◽

Iron Sulfur ◽

Natural Iron ◽

Lipoyl Synthase

Lipoyl synthase (LIAS) is an iron–sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe–4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron–sulfur (Fe–S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe–2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography–mass spectrometry (LC–MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS’s two [4Fe–4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe–4S] center. These results detail the trafficking of Fe–S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe–S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe–4S] cluster reconstitution is evident.

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Branched chain amino Acids as in vitro and in vivo Anti-Oxidation Compounds

Research Journal of Pharmacy and Technology ◽

10.52711/0974-360x.2021.00677 ◽

2021 ◽

pp. 3899-3904

Author(s):

Moath Alqaraleh ◽

Violet Kasabri ◽

Ibrahim Al-Majali ◽

Nihad Al-Othman ◽

...

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Amino Acids ◽

Branched Chain Amino Acids ◽

Raw 264.7 ◽

Raw 264.7 Cell ◽

Branched Chain ◽

Raw 264.7 Cell Line

Background and aims: Branched chain amino acids (BCAAs) can be tightly connected to metabolism syndrome (MetS) which can be counted as a metabolic indicator in the case of insulin resistance (IR). The aim of this study was to assess the potential role of these acids under oxidative stress. Material and Methods: the in vitro antioxidant activity of BCAAs was assessed using free radical 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) scavenging assays. For further check, a qRT-PCR technique was madefor detection the extent of alterations in gene expression of antioxidative enzymes (catalase and glutathione peroxidase (Gpx)) in lipopolysaccharides (LPS(-induced macrophages RAW 264.7 cell line. Additionally, BCAAs antioxidant activity was evaluated based on plasma H2O2 levels and xanthine oxidase (XO) activity in prooxidative LPS-treated mice. Results: Different concentrations of BCAAs affected on DPPH radical scavenging activity but to lesser extent than the ascorbic acid. Besides, BCAAs obviously upregulated the gene expression levels of catalases and Gpx in LPS-modulated macrophage RAW 264.7 cell line. In vivo BCAAs significantly minimized the level of plasma H2O2 as well as the activity of XO activity under oxidative stress. Conclusion: our current findings suggest that BCAAs supplementation may potentially serve as a therapeutic target for treatment of oxidative stress occurs with atherosclerosis, IR-diabetes, MetS and tumorigenesis.

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Production and utilization of amino acids by ovine placenta in vivo

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1998.274.1.e13 ◽

1998 ◽

Vol 274 (1) ◽

pp. E13-E22 ◽

Cited By ~ 22

Author(s):

Misoo Chung ◽

Cecilia Teng ◽

Michelle Timmerman ◽

Giacomo Meschia ◽

Frederick C. Battaglia

Keyword(s):

Amino Acids ◽

Branched Chain Amino Acids ◽

Plasma Amino Acids ◽

Physiological Conditions ◽

Ovine Placenta ◽

Branched Chain ◽

Glutamine Production

Uterine and umbilical uptakes of plasma amino acids were measured simultaneously in eighteen singleton pregnant ewes at 130 ± 1 days gestation for the purpose of establishing which amino acids are produced or used by the uteroplacenta under normal physiological conditions and at what rates. The branched-chain amino acids (BCAA) had uterine uptakes significantly greater than umbilical uptakes. Net uteroplacental BCAA utilization was 8.0 ± 2.5 μmol ⋅ kg fetus−1 ⋅ min−1( P < 0.005) and represented 42% of the total BCAA utilization by fetus plus uteroplacenta. There was placental uptake of fetal glutamate (4.2 ± 0.3 μmol ⋅ kg fetus−1 ⋅ min−1, P < 0.001) and no uterine uptake of maternal glutamate. Umbilical uptake of glutamine was ∼61% greater than uterine uptake, thus demonstrating net uteroplacental glutamine production of 2.2 ± 0.9 μmol ⋅ kg fetus−1 ⋅ min−1( P < 0.021). In conjunction with other evidence, these data indicate rapid placental metabolism of glutamate, which is in part supplied by the fetus and in part produced locally via BCAA transamination. Most of the glutamate is oxidized, and some is used to synthesize glutamine, which is delivered to the fetus. There was net uteroplacental utilization of maternal serine and umbilical uptake of glycine produced by the placenta. Maternal serine utilization and glycine umbilical uptake were virtually equal (3.14 ± 0.50 vs. 3.10 ± 0.46 μmol ⋅ kg fetus−1 ⋅ min−1). This evidence supports the conclusion that the ovine placenta converts large quantities of maternal serine into fetal glycine.

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Molecular Mechanisms Underlying the Positive Stringent Response of the Bacillus subtilis ilv-leu Operon, Involved in the Biosynthesis of Branched-Chain Amino Acids

Journal of Bacteriology ◽

10.1128/jb.00606-08 ◽

2008 ◽

Vol 190 (18) ◽

pp. 6134-6147 ◽

Cited By ~ 33

Author(s):

Shigeo Tojo ◽

Takenori Satomura ◽

Kanako Kumamoto ◽

Kazutake Hirooka ◽

Yasutaro Fujita

Keyword(s):

Amino Acids ◽

Bacillus Subtilis ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Stringent Response ◽

Branched Chain Amino Acids ◽

Base Substitution ◽

Branched Chain

ABSTRACT Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5′ end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

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Enzymic determination of branched-chain amino acids and 2-oxoacids in rat tissues. Transfer of 2-oxoacids from skeletal muscle to liver in vivo

Biochemical Journal ◽

10.1042/bj1880705 ◽

1980 ◽

Vol 188 (3) ◽

pp. 705-713 ◽

Cited By ~ 86

Author(s):

G Livesey ◽

P Lund

Keyword(s):

Skeletal Muscle ◽

Amino Acids ◽

Bacillus Subtilis ◽

Branched Chain Amino Acids ◽

Rat Tissues ◽

Arterial Blood ◽

Branched Chain ◽

Arteriovenous Difference

1. A procedure is described for the purification of leucine dehydrogenase (EC 1.4.1.9) from Bacillus subtilis. 2. The preparation is suitable for the quantitative assay of branched-chain amino acids and their 2-oxoacid analogues. 3. The content of total branched-chain 2-oxoacids in freeze-clamped liver, kidney, heart or mammary gland of fed rats is less than 5 nmol/g fresh wt. Higher amounts are present in skeletal muscle and arterial blood (25 +/- 4 nmol per g fresh wt., and 33 +/- 6 nmol per ml respectively; means +/- S.D. of 3 and 11 animals respectively). The values are not significantly affected by starvation for 24 h. 4. Arteriovenous difference measurements show that considerable amounts of branched-chain 2-oxoacids are released by skeletal muscle into the circulation and similar amounts are removed by the liver (about 1 mmol/24 h in a 400 g rat).

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Amino acid infusion increases the sensitivity of muscle protein synthesis in vivo to insulin. Effect of branched-chain amino acids

Biochemical Journal ◽

10.1042/bj2540579 ◽

1988 ◽

Vol 254 (2) ◽

pp. 579-584 ◽

Cited By ~ 209

Author(s):

P J Garlick ◽

I Grant

Keyword(s):

Amino Acids ◽

Protein Synthesis ◽

Amino Acid ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Branched Chain Amino Acids ◽

Acid Infusion ◽

Amino Acid Infusion ◽

Branched Chain

Rates of muscle protein synthesis were measured in vivo in tissues of post-absorptive young rats that were given intravenous infusions of various combinations of insulin and amino acids. In the absence of amino acid infusion, there was a steady rise in muscle protein synthesis with plasma insulin concentration up to 158 mu units/ml, but when a complete amino acids mixtures was included maximal rates were obtained at 20 mu units/ml. The effect of the complete mixture could be reproduced by a mixture of essential amino acids or of branched-chain amino acids, but not by a non-essential mixture, alanine, methionine or glutamine. It is concluded that amino acids, particularly the branched-chain ones, increase the sensitivity of muscle protein synthesis to insulin.

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Alteration in gene expression of branched-chain keto acid dehydrogenase kinase but not in gene expression of its substrate in the liver of clofibrate-treated rats

Biochemical Journal ◽

10.1042/bj3170411 ◽

1996 ◽

Vol 317 (2) ◽

pp. 411-417 ◽

Cited By ~ 22

Author(s):

Harbhajan S. PAUL ◽

Wei-Qun LIU ◽

Siamak A. ADIBI

Keyword(s):

Gene Expression ◽

Rat Liver ◽

Fold Increase ◽

Branched Chain Amino Acids ◽

The Other ◽

Mrna Levels ◽

Keto Acid ◽

Protein Mass ◽

Acid Dehydrogenase ◽

Branched Chain

We previously showed that the oxidation of branched-chain amino acids is increased in rats treated with clofibrate [Paul and Adibi (1980) J. Clin. Invest. 65, 1285–1293]. Two subsequent studies have reported contradictory results regarding the effect of clofibrate treatment on gene expression of branched-chain keto acid dehydrogenase (BCKDH) in rat liver. Furthermore, there has been no previous study of the effect of clofibrate treatment on gene expression of BCKDH kinase, which regulates the activity of BCKDH by phosphorylation. The purpose of the present study was to investigate the above issues. Clofibrate treatment for 2 weeks resulted in (a) a 3-fold increase in the flux through BCKDH in mitochondria isolated from rat liver, and (b) a modest but significant increase in the activity of BCKDH. However, clofibrate treatment had no significant effect on the mass of E1α, E1β, and E2 subunits of BCKDH or the abundance of mRNAs encoding these subunits. On the other hand, clofibrate treatment significantly reduced the activity, the protein mass and the mRNA levels of BCKDH kinase in the liver. In contrast to the results obtained in liver, clofibrate treatment had no significant effect on any of these parameters of BCKDH kinase in the skeletal muscle. In conclusion, our results show that clofibrate treatment increases the activity of BCKDH in the liver and the mechanism of this effect is the inhibition of gene expression of the BCKDH kinase.

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