Purified RecombinantEscherichia coliKetol-Acid Reductoisomerase Is Unsuitable for Use in a Coupled Assay of Acetohydroxyacid Synthase Activity due to an Unexpected Side Reaction

Hill, Craig; Duggleby, Ronald G.

doi:10.1006/prep.1998.0988

Cited by 12 publications

(16 citation statements)

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“…Therefore, we expressed such mutants using the E. coli host strain CU505 in which the ilv GMEDA and ilv YC operons are deleted. Because this strain does not contain the T7 RNA polymerase gene, we cloned the KARI gene ( ilv C) from our usual expression vector pET‐C [7] into a different vector, pProExHT, where expression is under the control of the lac promoter. The protein expressed by this vector has an N‐terminal hexahistidine tag, and it was purified in the same way as that expressed by pET‐C.…”

Section: Expression and Purificationmentioning

confidence: 99%

“…Wildtype hexahistidine‐tagged recombinant E. coli KARI was expressed using the plasmid pET‐C, which contains the ilv C gene that encodes KARI, cloned into the pET30a(+) plasmid [7]. Expression is under the control of the T7 promoter and therefore requires a host cell containing the T7 RNA polymerase gene.…”

Section: Enzyme Expression Purification and Mutagenesismentioning

confidence: 99%

“…We used the E. coli strain BL21 (DE3) for this purpose. The expressed enzyme has an N‐terminal hexahistidine tag and was purified by immobilized metal affinity chromatography as described previously [7]. The purified enzyme has a specific activity of ≈ 2 U·mg −1 when 2 m m 2‐acetolactate is used as the substrate.…”

Section: Enzyme Expression Purification and Mutagenesismentioning

confidence: 99%

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Probing the mechanism of the bifunctional enzyme ketol‐acid reductoisomerase by site‐directed mutagenesis of the active site

et al. 2004

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Ketol‐acid reductoisomerase (EC 1.1.1.86) is involved in the biosynthesis of the branched‐chain amino acids. It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site; an isomerization consisting of an alkyl migration, followed by an NADPH‐dependent reduction of a 2‐ketoacid. The 2‐ketoacid formed by the alkyl migration is not released. Using the pure recombinant Escherichia coli enzyme, we show that the isomerization reaction has a highly unfavourable equilibrium constant. The reductase activity is shown to be relatively nonspecific and is capable of utilizing a variety of 2‐ketoacids. The active site of the enzyme contains eight conserved polar amino acids and we have mutated each of these in order to dissect their contributions to the isomerase and reductase activities. Several mutations result in loss of the isomerase activity with retention of reductase activity. However, none of the 17 mutants examined have the isomerase activity only. We suggest a reason for this, involving direct reduction of a transition state formed during the isomerization, which is necessitated by the unfavourable equilibrium position of the isomerization. Our mechanism explains why the two activities must occur in a single active site without release of a 2‐ketoacid and provides a rationale for the requirement for NADPH by the isomerase.

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Section: Expression and Purificationmentioning

confidence: 99%

Section: Enzyme Expression Purification and Mutagenesismentioning

confidence: 99%

Section: Enzyme Expression Purification and Mutagenesismentioning

confidence: 99%

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Probing the mechanism of the bifunctional enzyme ketol‐acid reductoisomerase by site‐directed mutagenesis of the active site

et al. 2004

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“…We have described previously the cloning of the gene into the pET30a(+) vector [15]. The pET-C (6879 bp) plasmid was used as the template.…”

Section: Methodsmentioning

confidence: 99%

A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step

2004

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BackgroundSite-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR.ResultsThe strategy relies on the use of a limiting concentration of one of the flanking primers (reverse or forward) along with the normal concentration of mutagenic primer, plus a prolonged final extension cycle in the first PCR amplification step. This first round of PCR generates a megaprimer that is used subsequently in the second round of PCR, along with the second flanking primer, but without the intermediate purification of the megaprimer. The strategy has been used successfully with four different plasmids to generate various mutants.ConclusionThis strategy provides a very rapid, inexpensive and efficient approach to perform site-directed mutagenesis. The strategy provides an alternative to conventional megaprimer based site-directed mutagenesis, which is based on an intermediate gel purification step. The strategy gives a high frequency of mutagenesis.

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“…KARI activity was measured with a continuous assay method [13], following the consumption of NADPH at 340 nm and 30°C. The assay solution contained 0.2 mmol/L NADPH, 1 mmol/L MgCl 2 , 0.1 mmol/L substrate (2-acetolactate), and inhibitors(1-8), in 0.1 mol/L phosphate buffer(pH 8.0).…”

Section: Biological Activitiesmentioning

confidence: 99%

The design, synthesis of amide KARI inhibitors and their biological activities

Wang

et al. 2009

Front. Chem. China

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Ketol-acid reductoisomerase(KARI) is a promising target for the design of herbicides yet there are only few reports on the molecular design of KARI inhibitors. In this paper, based on the reported 0.165 nm high resolution crystal structure of the spinach KARI complex, 279 molecules with low binding energy toward KARI were obtained from an MDL/ACD 3D database search using the program DOCK 4.0. According to the structural information of 279 molecules provided, some amide compounds have been designed and synthesized. The bioassay results show that most of these amides had inhibitory activity to rice KARI at a test concentration of 200 μg/mL. Among which eight amides, compounds 1 and 6 show 57.4% and 48.1% inhibitory activity to KARI. The herbicidal activities of these amides were further investigated on di-cotyledonous rape (Brassica campestris) and mono-cotyledonous barnyardgrass (Echinochloa crusgalli). Compounds 1 and 6 were more favorable than others and showed 52.0% and 72.6% inhibitory activity on rape root at 100 μg/mL concentration, respectively. These amides could be further optimized for finding more potent candidates.Ketol-acid reductoisomerase (KARI, EC 1.1.1.86), also known as acetohydroxy acid isomeroreductase (AHIR), is one of the key enzymes for the synthesis of branched-chain amino acids (isoleucine, leucine and valine). There are reviews proposing KARI as a herbicidal target [1]. Although the molecular design of potential KARI inhibitors is in accord with green herbicidal research, there are few reports of new KARI inhibitors except Hoe 704, IpOHA, 1,2,3-thiadiazoles and CPD derivatives, which are potent inhibitors of the enzyme only in vitro, but their activities in vivo as herbicides are weak [2][3][4].In our previous work [5], 279 molecules with low combining energy toward KARI were obtained by an MDL/ACD-3D database search using the DOCK 4.0 program based on the crystal structure of the KARI complex, which provides us plentiful structural information for our research. Many amides were found among the 279 molecules. In looking for novel potent KARI inhibitors, several types of amides were synthesized and their herbicidal activities both in vitro and in vivo were investigated. The novel structures are shown in Fig. 1. Experiments Apparatus and materialsThe DOCK (version 4.0) program was employed. The computational work was based on the 0.165 nm crystal structure of the spinach KARI complex (PDB code 1YVE) and finished on the SGI Indigo R10000 of the Shanghai . The MDL/ACD 3D database of 250000 organic compounds was searched for potential KARI inhibitors.Melting points were determined using a Taike X-4 apparatus and were uncorrected. The infrared spectra were recorded on a BRUCK EQUINOX55 FTIR spectrophotometer as KBr tablets. 1 H-NMR and 13 C-NMR spectra were measured on a Bruker AV-300 instrument (300 MHz) using TMS as an internal standard. Mass spectra were recorded on a Hewlett Packard G1800A and an HP-1100 LC/MS instruments. Elemental analyses were performed on a Yanaco MT-3CHN elemental ana...

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Purified RecombinantEscherichia coliKetol-Acid Reductoisomerase Is Unsuitable for Use in a Coupled Assay of Acetohydroxyacid Synthase Activity due to an Unexpected Side Reaction

Cited by 12 publications

References 11 publications

Probing the mechanism of the bifunctional enzyme ketol‐acid reductoisomerase by site‐directed mutagenesis of the active site

Probing the mechanism of the bifunctional enzyme ketol‐acid reductoisomerase by site‐directed mutagenesis of the active site

A new approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step

The design, synthesis of amide KARI inhibitors and their biological activities

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