Preliminary X-ray crystallographic studies of the catalytic subunit of<i>Escherichia coli</i>AHAS II with its cofactors

Niu, Xuhui; Liu, Xiang; Zhou, Yufan; Niu, Congwei; Xi, Zhen; Su, Xiao‐Dong

doi:10.1107/s1744309111008839

Cited by 7 publications

(5 citation statements)

References 12 publications

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“…The only crystal structure of an AHAS in the absence of herbicide is that of yeast AHAS [63]. There are no bacterial AHAS catalytic subunit structures resolved to date, even after the identification of AHAS as a possible antimicrobial target, with the exception of a recent study on preliminary crystallization of the catalytic subunit of E. coli AHAS II [66]. Several attempts made in our laboratories aiming to crystallize M. tuberculosis and H. influenza AHASs were unsuccessful and are still in the preliminary stages.…”

Section: Bacterial Ahassmentioning

confidence: 99%

Bacterial acetohydroxyacid synthase and its inhibitors – a summary of their structure, biological activity and current status

Gedi

Yoon

2012

The FEBS Journal

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Acetohydroxyacid synthase (anabolic AHAS; http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/2/1/6.html) is a thiamin diphosphate‐dependent enzyme that catalyzes the first step in the branched‐chain amino acid (BCAA) biosynthesis pathway. BCAAs are synthesized by plants, algae, fungi and bacteria, although not by animals. Thus, the enzymes of the BCAA biosynthetic pathway are potential targets in the development of herbicides, fungicides and antimicrobial compounds. Plant AHASs are well studied in this regard because specific plant AHAS inhibitors are considered to comprise the most potent herbicides. These inhibitors are also effective against bacterial AHASs, inhibit the growth of several bacterial strains and have little to no toxicity in mammals. This review provides an overview of bacterial AHASs with an update of the current status of AHAS inhibitors.

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Section: Bacterial Ahassmentioning

confidence: 99%

Bacterial acetohydroxyacid synthase and its inhibitors – a summary of their structure, biological activity and current status

Gedi

Yoon

2012

The FEBS Journal

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“…Complete and accurate understanding of the mechanisms involved in acetaldehyde formation demands more in-depth study of the reaction intermediates and more importantly crystal structure of enzyme. Other than a preliminary X-ray crystal structure for catalytic subunit of E. coli AHAS II [44] , so far, no crystal structure has been provided for any catalytic (large) subunit of AHAS [45] .…”

Section: Resultsmentioning

confidence: 99%

Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

Eram

2016

Biochemistry and Biophysics Reports

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Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium Thermotoga maritima has been purified and characterized (kcat ~100 s−1). It was found that the same enzyme also had the ability to catalyze the production of acetaldehyde and CO2 from pyruvate, an activity of pyruvate decarboxylase (PDC) at a rate approximately 10% of its AHAS activity. Compared to the catalytic subunit, reconstitution of the individually expressed and purified catalytic and regulatory subunits of the AHAS stimulated both activities of PDC and AHAS. Both activities had similar pH and temperature profiles with an optimal pH of 7.0 and temperature of 85 °C. The enzyme kinetic parameters were determined, however, it showed a non-Michaelis-Menten kinetics for pyruvate only. This is the first report on the PDC activity of an AHAS and the second bifunctional enzyme that might be involved in the production of ethanol from pyruvate in hyperthermophilic microorganisms.

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“…The enzyme activity was determined in the presence of different concentrations and types of branched-chain amino acids. In the enzyme reaction system, three branched-chain amino acids with different concentration gradients were added, and the final concentrations of the three amino acids were 10, 20, 30, 40, 50, 60, 70, and 80 mM when the three amino acids were added separately; meanwhile, the final concentrations of the three amino acids when the three amino acids were added simultaneously were 5,10,15,20,25,30,35,40, and 30 mM.…”

Section: Anti-feedback Inhibition Analysismentioning

confidence: 99%

“…AHAS is found in plants, fungi, bacteria, and archaea, but it is not present in animals or humans. It consists of large and small subunits that form a multimeric group, with the large subunit performing its catalytic function and containing the binding sites of thiamine diphosphate, flavin adenine dinucleotide, and magnesium (Mg 2+ ) [2,3] and the small subunit acting as a regulator, containing binding sites for branched-chain amino acids and enhancing catalytic subunit (CSU) activity [4,5]. The CSUs of prokaryotes and eukaryotes have relatively conserved amino acid sequences and peptide lengths, which results in their identical sizes (approximately 60 kDa) [6,7].…”

Section: Introductionmentioning

confidence: 99%

Cloning, Expression, Enzymatic Characterization and Mechanistic Studies of M13 Mutant Acetohydroxyacid Synthase That Rescues Valine Feedback Inhibition

Tan,

Gao,

et al. 2024

Fermentation

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Acetohydroxyacid synthase (AHAS) is a key enzyme in the first step of the branched-chain amino acid synthesis pathway, and the production of acetohydroxybutyrate from one molecule of 2-ketobutyric acid and one molecule of pyruvate. AHAS is inhibited by feedback from L-valine, L-leucine, and L-isoleucine, and the expression of ilvBN, the gene encoding AHAS, is regulated by all three branched-chain amino acids. A change in amino acids 20–22 on the regulatory subunit (M13 mutation) removes the feedback inhibition by valine. We cloned the gene encoding AHAS (ilvBN) into a vector and then transfected it into Escherichia coli BL21 for expression with targeted changes in amino acids 20–22 on the regulatory subunit, and then determined the activity of the mutated AHAS and its inhibitory effects on valine, isoleucine, and leucine. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Previous studies have suggested that the binding sites for the three branched-chain amino acids may be at the same variable center. We investigated the enzymatic properties of wild-type and mutant AHAS, modeled their crystal structures, and resolved the mechanism of feedback inhibition induced by mutant M13, which will be useful for continuing the modification of AHAS and the design of broad-spectrum herbicides.

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Preliminary X-ray crystallographic studies of the catalytic subunit ofEscherichia coliAHAS II with its cofactors

Cited by 7 publications

References 12 publications

Bacterial acetohydroxyacid synthase and its inhibitors – a summary of their structure, biological activity and current status

Bacterial acetohydroxyacid synthase and its inhibitors – a summary of their structure, biological activity and current status

Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

Cloning, Expression, Enzymatic Characterization and Mechanistic Studies of M13 Mutant Acetohydroxyacid Synthase That Rescues Valine Feedback Inhibition

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