Characterization of Acetohydroxyacid Synthase I from<i>Escherichia coli</i>K-12 and Identification of Its Inhibitors

Chien, Pham Ngoc; Moon, Ji‐Young; Cho, Jun‐Haeng; Lee, Soo-Jae; Park, Joon‐Shik; Kim, Dong-Eun; Park, Yoonkyung; Yoon, Moon‐Young

doi:10.1271/bbb.100496

Cited by 8 publications

(9 citation statements)

References 23 publications

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“…coli AHAS I90377.0–7.520.800.95 33 E . coli K-12AHAS I65377.51.504.15 34 Pseudomonas aeruginosa PA01AHAS65377.50.1214.20 ± 0.20 35 Mycobacterium tuberculosis ilvB1 (Rv3003c)68.335–407.02.802.76 ± 0.12 36 Shigella sonnei AHAS65377.50.128.01 37 a MW, molecular weight by SDS-PAGE. b The activity of each enzyme was determined under the optimal conditions with pyruvate as a substrate.…”

Section: Resultsmentioning

confidence: 99%

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate

Jia

Liu

Han

2017

Sci Rep

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Acetoin (3-hydroxy-2-butanone) is an important bio-based platform chemical with wide applications. In vitro enzyme catalysed synthesis exhibits great feasibility in the production of chemicals with high purity. In the present work, a synthetic pathway involving a two-step continuous reaction was constructed in vitro for acetoin production from pyruvate at improved temperature. Thermostable candidates, acetolactate synthase (coAHASL1 and coAHASL2 from Caldicellulosiruptor owensensis OL) and α-acetolactate decarboxylase (bsALDC from Bacillus subtilis IPE5-4) were cloned, heterologously expressed, and characterized. All the enzymes showed maximum activities at 65–70 °C and pH of 6.5. Enzyme kinetics analysis showed that coAHASL1 had a higher activity but lower affinity against pyruvate than that of coAHASL2. In addition, the activities of coAHASL1 and bsALDC were promoted by Mn2+ and NADPH. The cascade enzymatic reaction was optimized by using coAHASL1 and bsALDC based on their kinetic properties. Under optimal conditions, a maximum concentration of 3.36 ± 0.26 mM acetoin was produced from 10 mM pyruvate after reaction for 24 h at 65 °C. The productivity of acetoin was 0.14 mM h−1, and the yield was 67.80% compared with the theoretical value. The results confirmed the feasibility of synthesis of acetoin from pyruvate with a cell-free enzyme catalysed system at improved temperature.

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Section: Resultsmentioning

confidence: 99%

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate

Jia

Liu

Han

2017

Sci Rep

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“…The additional substituent groups in the scaffold include electron‐withdrawing groups (NO 2 , F, Cl or CH 3 ) on benzene; CH 3 , OCH 3 or SCH 3 groups on triazole; and OCH 3 or Cl on pyrimidine. In addition, these four compounds showed more potent inhibition than any other herbicide against most of the bacterial AHASs tested in our laboratory, except for S. sonnei AHAS, which had a very weak inhibition [30,91,92]. Despite the fact that triazolopyrimidines had very weak inhibition in bacterial AHASs, these modified/substituted analogues showed extremely potent inhibition.…”

Section: Inhibitorsmentioning

confidence: 98%

“…8), KHG20613 and KHG20616 (data not shown) [8]. KHG20612 also showed a strong inhibition potency against H. influenza , E. coli K‐12 isozyme I and S. sonnei AHASs and weakly inhibited B. anthracis AHAS [30,89,91]. With all the tested AHASs, KHG20612 showed better inhibition than any other tested herbicide, except in the case of B. anthracis AHAS.…”

Section: Inhibitorsmentioning

confidence: 99%

“…The discontinuous colourimetric method employs a high-throughput format where various chemical compound libraries are screened against several bacterial AHASs with the aim of developing antimicrobial agents. This strategy has been applied against the purified AHAS of several pathogenic bacteria, including M. tuberculosis, B. anthracis, H. influenza, P. aeruginosa, S. sonnei and E. coli K-12 isozyme I [8,30,44,89,91,92]. The screening identified several inhibitors, each representing a structural class, including 3-phenyl- [1,2,4]triazol-1-yl, substituted triazol-1-yl-pyrimidine derivatives and phenyl-2,3-dihydro-isothiazole derivatives [8,44,92].…”

Section: Other Compoundsmentioning

confidence: 99%

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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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“…AHAS from the enterobacteria E. coli is the most thoroughly investigated of all the AHASs from different organisms. Three isozymes, namely AHAS I, II and III have been characterized in this enteric bacterium and up to date, the individual subunits of each isozyme have been expressed and purified to homogeneity (18)(19)(20)(21)(22). It is noteworthy that AHAS I is not only the most active one of the three isozymes, but also one of the most potent enzymes catalyzing the formation of acetolactate amongst all the known AHASs of bacterial origin (1,21).…”

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confidence: 99%

Cloning and characterization of GST fusion tag stabilized large subunit of Escherichia coli acetohydroxyacid synthase I

Liu

Wang

et al. 2016

Journal of Bioscience and Bioengineering

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There are three acetohydroxyacid synthase (AHAS, EC 4.1.3.18) isozymes (I, II, and III) in the enterobacteria Escherichia coli among which AHAS I is the most active. Its large subunit (LSU) possesses full catalytic machinery, but is unstable in the absence of the small subunit (SSU). To get applicable LSU of AHAS I, we prepared and characterized in this study the polypeptide as a His-tagged (His-LSU) and a glutathione S-transferase (GST)-tagged (GST-LSU) fusion protein, respectively. The results showed that the His-LSU is unstable, whereas the GST-LSU displays excellent stability. This phenomenon suggests that the GST polypeptide fusion tag could stabilize the target protein when compared with histidine tag. It is the first time that the stabilizing effect of the GST tag was observed. Further characterization of the GST-LSU protein indicated that it possesses the basic functions of AHAS I with a specific activity of 20.8 μmol min(-1) mg(-1) and a Km value for pyruvate of 0.95 mM. These observations imply that introduction of the GST fusion tag to LSU of AHAS I does not affect the function of the protein. The possible reasons that the GST fusion tag could make the LSU stable are initially discussed.

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Characterization of Acetohydroxyacid Synthase I fromEscherichia coliK-12 and Identification of Its Inhibitors

Cited by 8 publications

References 23 publications

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate

A thermophilic cell-free cascade enzymatic reaction for acetoin synthesis from pyruvate

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

Cloning and characterization of GST fusion tag stabilized large subunit of Escherichia coli acetohydroxyacid synthase I

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