Biochemical and transcription analysis of acetohydroxyacid synthase isoforms in Mycobacterium tuberculosis identifies these enzymes as potential targets for drug development

Singh, Vinayak; Chandra, Deepak; Srivastava, Bhavana; Srivastava, Ranjana

doi:10.1099/mic.0.041343-0

Cited by 23 publications

(19 citation statements)

References 36 publications

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“…The function of AHAS was also confirmed in the synthetic pathway for BCAAs by genetic analysis 7 . An activity assay revealed that the catalytic subunits coAHASL1 and coAHASL2 had their maximum activity at 65–70 °C at a pH of 6.5, which was consistent with other reported catalytic subunits 25, 26 . The two catalytic subunits showed both ThDP- and FAD-dependent catalytic activity, which was in line with the characteristics for the AHAS catalytic subunit 27 .…”

Section: Discussionsupporting

confidence: 90%

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: Discussionsupporting

confidence: 90%

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

Jia

Liu

Han

2017

Sci Rep

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“…In the M. tuberculosis genome, four catalytic AHAS subunits ( ilv B1, Rv3003c; ilv B2, Rv3470c; ilv G, Rv1820; and ilv X, Rv3509c) and one regulatory subunit ( ilv N, Rv3002c) have been annotated and the genes are dispersed along the chromosome. 31,32 A putative small regulatory subunit has also been identified ( ilv H) and is believed to be the regulatory subunit of ilv B2, based on similarities with the E. coli enzymes. 33 The ilv B1– ilv N pair displays the enzymatic characteristics expected for the AHAS involved in the biosynthesis of BCAAs, including cofactor requirements.…”

Section: Ilvb/n Acetolactate (Acetohydroxyacid) Synthasementioning

confidence: 99%

Bacterial Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms, and Drugability

2017

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The eight enzymes responsible for the biosynthesis of the three branched-chain amino acids (l-isoleucine, l-leucine, and l-valine) were identified decades ago using classical genetic approaches based on amino acid auxotrophy. This review will highlight the recent progress in the determination of the three-dimensional structures of these enzymes, their chemical mechanisms, and insights into their suitability as targets for the development of antibacterial agents. Given the enormous rise in bacterial drug resistance to every major class of antibacterial compound, there is a clear and present need for the identification of new antibacterial compounds with nonoverlapping targets to currently used antibacterials that target cell wall, protein, mRNA, and DNA synthesis.

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“…Several anabolic AHASs from various organisms have been characterized including different isozymes (isozymes I, II, and III) from Escherichia coli [17] , [18] , [19] , and Salmonella [20] , Corynebacterium glutamicum [21] , Klebsiella pneumoniae [22] , different pathogenic mycobacteria species [23] , [24] , [25] , [26] , Shigella sonnei [27] and Haemophilus influenzae [28] , Saccharomyces cerevisiae [16] , and plants including Arabidopsis ( Arabidopsis thaliana ) [29] , and tobacco ( Nicotiana tabacum ) [30] . Considering the absence of the branched chain amino acid (BCAA) biosynthesis pathway in animals, the anabolic AHAS has spurred significant interest as a potential candidate for development of new antimicrobial drugs and herbicides (reviewed in Refs.…”

Section: Introductionmentioning

confidence: 99%

Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima

Eram

Sarafuddin

Gong

et al. 2015

Biochemistry and Biophysics Reports

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Acetohydroxyacid synthase (AHAS) is the key enzyme in branched chain amino acid biosynthesis pathway. The enzyme activity and properties of a highly thermostable AHAS from the hyperthermophilic bacterium Thermotoga maritima is being reported. The catalytic and regulatory subunits of AHAS from T. maritima were over-expressed in Escherichia coli. The recombinant subunits were purified using a simplified procedure including a heat-treatment step followed by chromatography. A discontinuous colorimetric assay method was optimized and used to determine the kinetic parameters. AHAS activity was determined to be present in several Thermotogales including T. maritima. The catalytic subunit of T. maritima AHAS was purified approximately 30-fold, with an AHAS activity of approximately 160±27 U/mg and native molecular mass of 156±6 kDa. The regulatory subunit was purified to homogeneity and showed no catalytic activity as expected. The optimum pH and temperature for AHAS activity were 7.0 and 85 °C, respectively. The apparent Km and Vmax for pyruvate were 16.4±2 mM and 246±7 U/mg, respectively. Reconstitution of the catalytic and regulatory subunits led to increased AHAS activity. This is the first report on characterization of an isoleucine, leucine, and valine operon (ilv operon) enzyme from a hyperthermophilic microorganism and may contribute to our understanding of the physiological pathways in Thermotogales. The enzyme represents the most active and thermostable AHAS reported so far.

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Biochemical and transcription analysis of acetohydroxyacid synthase isoforms in Mycobacterium tuberculosis identifies these enzymes as potential targets for drug development

Cited by 23 publications

References 36 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 Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms, and Drugability

Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima

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