Heterologous Expression and Functional Characterization of Catalytic Subunit of Rice Acetohydroxyacid Synthase

2019 ◽  
Vol 26 (3) ◽  
pp. 176-183
Author(s):  
Ghazaleh Arabzadeh ◽  
Azar Shahpiri
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Feedback Inhibition ◽  
Functional Characterization ◽  
Branched Chain Amino Acids ◽  
Biochemical Properties ◽  
Catalytic Subunit ◽  
Acetohydroxyacid Synthase ◽  
Branched Chain

Background: Acetohydroxyacid Synthase (AHAS) is the first enzyme in the biosynthesis pathway of the branched chain amino acids. AHAS is the common target site of five herbicide chemical groups: sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidinyl-thiobenzoates, and sulfonyl-aminocarbonyl-triazolinone. </P><P> Objective: The purification of protein enabled us to study the physical and biochemical properties of the enzyme. In addition in vitro activity of this enzyme was tested in the presence of four different sulfonylureaherbicides and the feedback regulation of enzyme was analyzed in the presence of branched amino acids. Methods: The gene encoding catalytic subunit of rice AHAS (cOsAHAS) without part of the chloroplast transit sequence was cloned into the bacterial expression vector pET41a and heterologously expressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST).The soluble protein was purified using affinity chromatography. The measurement of GSTOsAHAS activity was performed under optimized conditions at present of branched-chain amino acids and sulfonylurea herbicides independently. Results: The optimum pH and temperature for GST-cOsAHAS activity was 8.0 and 37 °C, respectively. The specific activity and Km value of this enzyme toward pyruvate were 0.08 U/mg and 30 mM, respectively.GST-cOsAHAS was inhibited by herbicides tribenuron, sulfosulfuron, nicosulfuron and bensulfuron while the enzyme was insensitivieto end products. Conclusion: These results suggest that the recombinant form of GST-cOsAHAS is functionally active and carries the binding site for sulfynylurea herbicides. Furthermore, GST-cOsAHAS was insensitive to feedback inhibition by endproducts which indicates the existence of a regulator subunit in rice AHAS as previously has been described in other plant AHASs.

scholarly journals Engineering Herbicide-Tolerance Rice Expressing an Acetohydroxyacid Synthase with a Single Amino Acid Deletion

2020 ◽  
Vol 21 (4) ◽  
pp. 1265
Author(s):  
Jun Fang, ◽  
Changzhao Wan ◽  
Wei Wang ◽  
Liuyin Ma ◽  
Xinqi Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Agronomic Traits ◽  
Herbicide Tolerance ◽  
Branched Chain Amino Acids ◽  
Single Amino Acid ◽  
Acetohydroxyacid Synthase ◽  
Sulfometuron Methyl ◽  
Herbicide Tolerant ◽  
Branched Chain

The acetohydroxyacid synthase (AHAS) is an essential enzyme involved in branched amino acids. Several herbicides wither weeds via inhibiting AHAS activity, and the AHAS mutants show tolerance to these herbicides. However, most AHAS mutations are residue substitutions but not residue deletion. Here, residue deletion was used to engineering the AHAS gene and herbicide-tolerant rice. Molecular docking analysis predicted that the W548 of the AHAS was a residue deletion to generate herbicide tolerance. The AHAS-ΔW548 protein was generated in vitro to remove the W548 residue. Interestingly, the deletion led to the tetramer dissociation of the AHAS, while this dissociation did not reduce the activity of the AHAS. Moreover, the W548 deletion contributed to multi-family herbicides tolerance. Specially, it conferred more tolerance to sulfometuron-methyl and bispyribac-sodium than the W548L substitution. Further analysis revealed that AHAS-ΔW548 had the best performance on the sulfometuron-methyl tolerance compared to the wild-type control. Over-expression of the AHAS-ΔW548 gene into rice led to the tolerance of multiple herbicides in the transgenic line. The T-DNA insertion and the herbicide treatment did not affect the agronomic traits and yields, while more branched-chain amino acids were detected in transgenic rice seeds. Residue deletion of W548 in the AHAS could be a useful strategy for engineering herbicide tolerant rice. The increase of branched-chain amino acids might improve the umami tastes of the rice.

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

2021 ◽  
pp. 3899-3904
Author(s):  
Moath Alqaraleh ◽  
Violet Kasabri ◽  
Ibrahim Al-Majali ◽  
Nihad Al-Othman ◽  
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.

A1151 EFFECT OF BRANCHED CHAIN AMINO ACIDS (BCAA) ON DIAPHRAGM FATIGUE IN VITRO

1990 ◽  
Vol 73 (3A) ◽  
pp. NA-NA
Author(s):  
H. Yamada ◽  
Y. Ohta ◽  
I. Chaudhry ◽  
H. Nagashima ◽  
J. Askanazi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Branched Chain Amino Acids ◽  
Branched Chain ◽  
Diaphragm Fatigue

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

2008 ◽  
Vol 190 (18) ◽  
pp. 6134-6147 ◽  
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.

Rapid Germination of Sulfonylurea-ResistantKochia scopariaL. Accessions is Associated with Elevated Seed Levels of Branched Chain Amino Acids

Weed Science ◽  
1993 ◽  
Vol 41 (1) ◽  
pp. 18-22 ◽  
Author(s):  
William E. Dyer ◽  
Peng W. Chee ◽  
Peter K. Fay
Keyword(s):  
Amino Acids ◽  
Thermal Gradient ◽  
Feedback Inhibition ◽  
Acetolactate Synthase ◽  
Branched Chain Amino Acids ◽  
Resistant Accession ◽  
Early Germination ◽  
Percent Germination ◽  
Soil Temperatures ◽  
Branched Chain

Field observations indicate that sulfonylurea-resistant kochia may germinate at lower soil temperatures and/or germinate more rapidly than susceptible kochia in the absence of herbicide. To investigate this possibility, seeds from three resistant and two susceptible kochia accessions were germinated at temperatures ranging from 4.6 to 13.2 C on thermal gradient plates. At 4.6 and 13.2 C, germination rates of all resistant accessions were higher than susceptible accessions, while germination rates of one resistant accession were higher than susceptible accessions at 7.2 and 10.5 C. Percent germination of all resistant accessions was significantly higher than susceptible accessions after 48 h at 4.6 C. At higher temperatures, percent germination of some resistant accessions was higher after 12 or 24 h, but germination of all accessions was similar at later times. HPLC analysis revealed that seeds from resistant accessions contained about 2-fold higher free levels of branched chain amino acids than seeds from susceptible accessions. The results indicate that mutations conferring resistance to sulfonylurea herbicides in these kochia accessions may concomitantly reduce or abolish acetolactate synthase sensitivity to normal feedback inhibition patterns, resulting in elevated levels of branched chain amino acids available for cell division and growth during early germination.

scholarly journals In Vitro Transcriptional Studies of thebkd Operon of Pseudomonas putida:l-Branched-Chain Amino Acids and d-Leucine Are the Inducers

1999 ◽  
Vol 181 (9) ◽  
pp. 2889-2894 ◽  
Author(s):  
Kunapuli T. Madhusudhan ◽  
Jinhe Luo ◽  
John R. Sokatch
Keyword(s):  
Amino Acids ◽  
Transcriptional Activation ◽  
In Vitro Transcription ◽  
Branched Chain Amino Acids ◽  
Multienzyme Complex ◽  
Keto Acids ◽  
Transcription In Vitro ◽  
Branched Chain ◽  
Hydroxyl Radical Footprinting

ABSTRACT BkdR is the transcriptional activator of the bkdoperon, which encodes the four proteins of the branched-chain keto acid dehydrogenase multienzyme complex of Pseudomonas putida. In this study, hydroxyl radical footprinting revealed that BkdR bound to only one face of DNA over the same region identified in DNase I protection assays. Deletions of even a few bases in the 5′ region of the BkdR-binding site greatly reduced transcription, confirming that the entire protected region is necessary for transcription. In vitro transcription of the bkd operon was obtained by using a vector containing the bkdR-bkdA1 intergenic region plus the putative ρ-independent terminator of the bkdoperon. Substrate DNA, BkdR, and any of thel-branched-chain amino acids or d-leucine was required for transcription. Branched-chain keto acids,d-valine, and d-isoleucine did not promote transcription. Therefore, the l-branched-chain amino acids and d-leucine are the inducers of the bkdoperon. The concentration of l-valine required for half-maximal transcription was 2.8 mM, which is similar to that needed to cause half-maximal proteolysis due to a conformational change in BkdR. A model for transcriptional activation of the bkdoperon by BkdR during enzyme induction which incorporates these results is presented.

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