Acetohydroxyacid Synthase Activity Research Articles

Re-investigation of in vitro activity of acetohydroxyacid synthase I holoenzyme from Escherichia coli

Acetohydroxyacid synthase (AHAS) is one of the key enzymes of the biosynthesis of branched-chain amino acids, it is also an effective target for the screening of herbicides and antibiotics. In this study we present a method for preparing Escherichia coli AHAS I holoenzyme (EcAHAS I) with exceptional stability, which provides a solid ground for us to re-investigate the in vitro catalytic properties of the protein. The results show EcAHAS I synthesized in this way exhibits similar function to Bacillus subtilis acetolactate synthase in its catalysis with pyruvate and 2-ketobutyrate (2-KB) as dual-substrate, producing four 2-hydroxy-3-ketoacids including (S)-2-acetolactate, (S)-2-aceto-2-hydroxybutyrate, (S)-2-propionyllactate, and (S)-2-propionyl-2-hydroxybutyrate. Quantification of the reaction indicates that the two substrates almost totally consume, and compound (S)-2-aceto-2- hydroxybutyrate forms in the highest yield among the four major products. Moreover, the protein also condenses two molecules of 2-KB to furnish (S)-2-propionyl-2-hydroxybutyrate. Further exploration manifests that EcAHAS I ligates pyruvate/2-KB and nitrosobenzene to generate two arylhydroxamic acids N-hydroxy-N-phenylacetamide and N-hydroxy-N-phenyl- propionamide. These findings enhance our comprehension of the catalytic characteristics of EcAHAS I. Furthermore, the application of this enzyme as a catalyst in construction of C–N bonds displays promising potential.

Efficient production of isobutanol from glycerol in Klebsiella pneumoniae: Regulation of acetohydroxyacid synthase, a rate-limiting enzyme in isobutanol biosynthesis

Glycerol is an abundant and inexpensive resource that can be used to produce valuable industrial products. Isobutanol is an important industrial chemical that has been studied for biosynthesis from various carbon sources and microorganisms. So far, isobutanol production by Klebsiella pneumoniae has mainly been studied using glucose. In this study, we produced isobutanol from glycerol based on the K. pneumoniae ΔldhAΔbudA mutant harboring pBR-iBO, which was used in a previous study with K. pneumoniae. We investigated the effect of different acetohydroxyacid synthase (AHAS) isoenzymes (rate-limiting enzymes in isobutanol biosynthesis), plasmid copy number, and different promoters as a method to increase isobutanol production through regulation at gene expression level. The K. pneumoniae Cu ΔldhAΔbudA, pUC-tac-BN-ISO strain produced 2.56-fold more isobutanol than reported previously for glycerol-derived isobutanol production. Additionally, the in vitro enzyme activity of AHAS I (ilvBN) was greater than that of the other two isoenzymes (ilvIH and ilvGM). The evaluation of process factors indicated that an agitation speed of 200 rpm with the culture maintained at pH 6.0 was a favorable condition for isobutanol production (1.02 g/L). Our results demonstrated enhanced production of isobutanol from glycerol and provide insights for future research on isobutanol production from renewable feedstock.

Seed imbibition by imazamox induces activation of glutathione S-transferases (GSTs) in wheat seedlings

Imidazolinone herbicides are widely used in agriculture. In some cases, they may be very persistent in soils, generating risks for the next crop in crop rotation. In several plant species, glutathione S-transferases (GSTs) take part in the detoxification of imazamox, but there exist no reports about their activity in wheat. Our study evaluated the effects of the imbibition of wheat seeds in imazamox containing solutions (10, 25 and 50 µM) on seedling growth, protein content as well as on the activity of GSTs. Growth of the young wheat seedlings was significantly inhibited after imbibition of the seeds in imazamox containing solutions. The protein content of both roots and shoots was lower compared to controls. This was partially due to a lower acetohydroxy acid synthase (AHAS) activity, leading to slowed protein turnover. The GSTs activity, determined using two different substrates 1-Chloro-2,4-dinitrobenzene (CDNB) and p-nitrophenyl acetate (pNPA), was enhanced, indicating their possible participation in cellular defence mechanisms against imazamox. The obtained new information about GSTs will be further verified in our subsequent studies.

An integrated approach for the characterization of one‐ and two‐gene imazamox‐resistant wheat lines

AbstractIn wheat (Triticum aestivum L.), cultivars resistant to imidazolinone herbicides are an important tool for weed management. The target site of imidazolinones is acetohydroxyacid synthase (AHAS), an enzyme coded by three homeologous genes (ahasL‐A1, ahasL‐B1, and ahasL‐D1). This study aimed to evaluate the effect of the imidazolinone herbicide imazamox on a single‐gene (AhasL‐D1) resistant line (R1) and a two‐gene (AhasL‐B1 and AhasL‐D1) resistant line (R2) at multiple biological levels. These lines showed variation in the transcriptional levels of ahasL homeologs, but no differences in total AHAS activity. The relative contribution of resistant alleles to the ahasL transcript pool accounted for 47% in the R1 line and 77% in the R2 line. The in vitro AHAS inhibition of the R1 line was intermediate between the R2 and the susceptible lines. At an anatomical level, high imazamox concentrations induced alterations in the root tip features of R1 plants but did not affect R2 plants, whereas at in vivo AHAS activity and whole‐plant levels, both resistant lines showed high resistance. The herbicide dose that reduced shoot biomass of resistant lines by 50% was 35‐fold higher than that of the susceptible line. We conclude that homeolog variation allows a higher contribution of resistance genes to the ahasL transcript pool in the R1 and R2 lines. Although both resistant lines showed high whole‐plant resistance, the differential root damage induced by imazamox between wheat lines suggests that two‐gene resistant cultivars could be an effective option to avoid the negative impacts caused by herbicide residues in the soil.

Imazamox detoxification and recovery of plants after application of imazamox to an imidazolinone resistant sunflower hybrid

Imidazolinone herbicides combined with imidazolinone resistant (IMI-R) crops provide a tool for solving the important problem of the occurrence of weeds during the early growth stages of sunflower. These herbicides inhibit the synthesis of branched chain amino acids by interrupting the key enzyme acetohydroxyacid synthase (AHAS). We studied the imazamox detoxification in an IMI-R sunflower hybrid together with plant growth and photosynthetic performance. Inhibition of photosynthesis and growth were observed as initial effects of imazamox application. A slight decrease in AHAS activity was also noticed. These effects disappeared within two weeks after application. A fast and well-functioning detoxification mechanism for the herbicide, of which the content decreased for about 90 % at 14 d after application, seems to be responsible for this. The activity of the xenobiotic detoxifying enzyme glutathione S-transferases (GSTs) significantly increased after imazamox application. Our results suggest that the metabolite glutathione serves as an auxiliary tool for imazamox detoxification through conjugation reactions realized by the GSTs, thereby taking part in the non-target mechanisms of resistance in IMI-R sunflower hybrids.

Variation in mutations providing resistance to acetohydroxyacid synthase inhibitors in Cyperus difformis in China

Cyperus difformis has evolved resistance to pyrazosulfuron-ethyl and other acetohydroxyacid synthase (AHAS) inhibitors in paddy fields in China. To understand the distribution of resistance and the mutations involved, 38 populations collected were from 7 provinces and compared. Application of pyrazosulfuron-ethyl at 30 g a.i. ha−1 identified 16 populations that survived, demonstrating resistance to this herbicide. Two exons of 498 and 1428 bp in length and a 1228–1233-bp intron of AHAS were cloned by genome walking, and three pairs of primers were designed to amplify eight conserved regions in this gene. In the 16 resistant (R) populations, five different types of mutations in the conserved region of the AHAS gene were identified: Pro-197-Ser, Pro-197-Arg, Pro-197-Leu, Asp-376-Glu, and Trp-574-Leu. Three R populations, YX15-22, YX12-10 and YX15-38, were chosen for in vitro AHAS activity assays, and the results showed that AHAS from YX15–22 carrying the Pro-197 mutation was insensitive to pyrazosulfuron-ethyl (resistance index (RI) = 310.0) and penoxsulam (RI = 10.0), whereas the enzyme from YX12–-10 and YX15-38 was insensitive to pyrazosulfuron-ethyl, penoxsulam, imazapic and bispyribac‑sodium (RI values ranging from 4.3 to 4462.0). AHAS inhibitor cross-resistance bioassays showed that YX12-10 and YX15-38 had cross-resistance to all of the tested herbicides (RI values ranging from 5.8 to 3321.9), while the YX15–22 population only had resistance to pyrazosulfuron-ethyl (RI = 827.4) and penoxsulam (RI = 6.6). This study clarified the distribution of resistant C. difformis in China and the different cross-resistance patterns given by various mutation types of AHAS.

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

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.

The 138th residue of acetohydroxyacid synthase in Corynebacterium glutamicum is important for the substrate binding specificity

Corynebacterium glutamicum acetohydroxyacid synthase (AHAS), composed of two subunits IlvB and IlvN, catalyzes the first reaction in the biosynthetic pathway of branched-chain amino acids. It either condenses two pyruvates to yield acetolactate, leading to the biosynthesis of L-valine and L-leucine, or condenses pyruvate with 2-ketobutyrate to yield acetohydroxybutyrate, leading to L-isoleucine biosynthesis. However, the mechanism for the substrate specificity of C. glutamicum AHAS remains unknown. In this study, AHASs from an L-valine-producing C. glutamicum VWB-1 and an L-isoleucine-producing C. glutamicum IWJ001 were analyzed. The amino acid sequence of IlvN from both strains are the same, but the 138th and 404th residues of IlvB from the two strains are different; they are alanine and valine in IWJ001 (IlvB138A404V), but valine and alanine in VWB-1 (IlvB138V404A). When IlvB138A404V and IlvB138V404A were overexpressed in wild type C. glutamicum ATCC14067 and its △alr△aceE△ilvA△leuA mutant YTW-104, the latter led to much more L-valine production than the former. AHAS activity studies also showed that the 138th valine was important for binding the 2nd substrate pyruvate but not the 404th alanine. YTW-104/pJYW4-ilvB138V404A-ilvNCE could produce 25.93 g/L L-valine. The results indicate that the 138th valine of IlvB in AHAS could play an important role, leading to the increased L-valine biosynthesis in C. glutamicum.

Transcript Profiling of Non‐Target‐Site Imidazolinone Resistance in Imisun Sunflower

Imidazolinone resistance found in a wild sunflower (Helianthus annuus L.) population was successfully transferred to a cultivated inbred line developing ‘Imisun’ sunflowers. Genetic regulation of this trait has been reported to involve two genes: Imr1, an allelic variant of ahasl1 locus that codes for acetohydroxyacid synthase catalytic subunit, and the modifier Imr2, whose identity remains unknown, but it could be related to non‐target‐site resistance such as xenobiotic metabolism. The aim of the present study was to characterize the gene expression of resistant and susceptible sunflower lines in response to imazethapyr herbicide by complementary DNA amplified fragment‐length polymorphism (cDNA‐AFLP). Three assays were performed to determine (i) optimal herbicide treatment concentration, (ii) duration of herbicide treatment, and (iii) in vitro acetohydroxyacid synthase activity to assess enzyme inhibition levels. An important number of genes related to metabolism of xenobiotics and stress was found: cytochrome P450 monooxygenases, UDP‐glucuronosyl/UDP‐glucosyltransferases, glycosyltransferases, and ATP‐binding cassette transporters, among others. These results suggest that non‐target‐site resistance mechanisms may contribute to herbicide resistance in Imisun sunflower and could be related to the modifier gene Imr2. Using cDNA‐AFLP, we were able to detect candidate detoxification‐related genes potentially involved in imidazolinone resistance in sunflower.

Male Sterility of an AHAS-Mutant Induced by Tribenuron-Methyl Solution Correlated With the Decrease of AHAS Activity in Brassica napus L.

Tribenuron-methyl (TBM), an acetohydroxyacid synthase (AHAS)-inhibiting herbicide, can be used as an efficient chemical hybridization agent to induce male sterility for practical utilization of heterosis in rapeseed (Brassica napus L.). Utilization of rapeseed mutants harboring herbicide-resistant AHAS alleles as the male parent can simplify the hybrid seed production protocol. Here we characterized a novel TBM-resistant mutant K5 derived from an elite rapeseed variety, Zhongshuang No. 9 (ZS9), by ethyl methyl sulfonate mutagenesis. Comparative analysis of three BnAHAS genes (BnAHAS1, BnAHAS2, and BnAHAS3) between the mutant K5 and ZS9 identified a C-to-T transition at 544 from the translation start site in BnAHAS1 in K5 (This resistant allele is referred to as BnAHAS1544T), which resulted in a substitution of proline with serine at 182 in BnAHAS1. Both ZS9 and K5 plants could be induced complete male sterility under TBM treatment (with 0.10 and 20 mg⋅L-1 of TBM, respectively). The relationship between TBM-induced male sterility (Y) and the relative AHAS activity of inflorescences (X) could be described as a modified logistic function, Y = 100-A/(1+Be(-KX)) for the both genotypes, although the obtained constants A, B, and K were different in the functions of ZS9 and K5. Transgenic Arabidopsis plants expressing BnAHAS1544T exhibited a higher TBM resistance of male reproductive organ than wild type, which confirmed that the Pro-182-Ser substitution in BnAHAS1 was responsible for higher TBM-resistance of male reproductive organs. Taken together, our findings provide a novel valuable rapeseed mutant for hybrid breeding by chemical hybridization agents and support the hypothesis that AHAS should be the target of the AHAS-inhibiting herbicide TBM when it is used as chemical hybridization agent in rapeseed.

Seasonal variation in acetohydroxy acid synthase inhibition by imazapyr in Cynodon dactylon

Cynodon dactylon (cynodon) is a weed that is particularly difficult to control due to its ability to reproduce readily using stolons and rhizomes. Although glyphosate is the most commonly used herbicide to control cynodon, current recommendations involve repetitive applications, which appear likely to select for resistance. Rotating glyphosate with herbicides that have different modes of action will be a more sustainable strategy for cynodon control. Earlier work suggested that the growth of cynodon is strongly inhibited by imazapyr, an herbicide that inhibits the acetohydroxy acid synthase (AHAS) enzyme. The optimum time for application of imazapyr on cynodon, and the subsequent inhibition of AHAS activity in cynodon are unknown. The aims of this study were firstly, to determine the effectiveness of inhibiting AHAS activity when applied at different times of the year, and secondly to compare the efficacy of foliar and root applications. Results showed that application time has a significant effect on the efficacy of imazapyr to inhibit enzyme activity, with mid–late summer being optimal, and furthermore that the herbicide can be absorbed by both the shoots and the roots.

High-level production of valine by expression of the feedback inhibition-insensitive acetohydroxyacid synthase in Saccharomyces cerevisiae

Valine, which is one of the branched-chain amino acids (BCAAs) essential for humans, is widely used in animal feed, dietary supplements and pharmaceuticals. At the commercial level, valine is usually produced by bacterial fermentation from glucose. However, valine biosynthesis can also proceed in the yeast Saccharomyces cerevisiae, which is a useful microorganism in fermentation industry. In S. cerevisiae, valine biosynthesis is regulated by valine itself via the feedback inhibition of acetohydroxyacid synthase (AHAS), which consists of two subunits, the catalytic subunit Ilv2 and the regulatory subunit Ilv6. In this study, to improve the valine productivity of yeast cells, we constructed several variants of Ilv6 by introducing amino acid substitutions based on a protein sequence comparison with the AHAS regulatory subunit of E. coli. Among them, we found that the Asn86Ala, Gly89Asp and Asn104Ala variants resulted in approximately 4-fold higher intracellular valine contents compared with those in cells with the wild-type Ilv6. The computational analysis of Ilv6 predicted that Asn86, Gly89 and Asn104 are located in the vicinity of a valine-binding site, suggesting that amino acid substitutions at these positions induce conformational change of the valine-binding site. To test the effects of these variants on AHAS activity, both recombinant Ilv2 and Ilv6 were purified and reconstituted in vitro. The Ilv6 variants were much less sensitive to feedback inhibition by valine than the wild-type Ilv6. Only a portion of the amino acid changes identified in the E. coli AHAS regulatory subunit IlvH enhanced the valine synthesis, suggesting structural and/or functional differences between the S. cerevisiae and E. coli AHAS regulatory subunits. It should also be noted that these amino acid substitutions did not affect the intracellular pools of the other BCAAs, leucine and isoleucine. The approach described here could be a practical method for the development of industrial yeast strains with high-level production of valine or isobutanol.

AHAS Trp574Leu substitution in Raphanus sativus L.: screening, enzyme activity and fitness cost.

Feral radish (Raphanus sativus L.) is a problematic weed that has become resistant to acetohydroxyacid synthase (AHAS) inhibitor herbicides due to the Trp574Leu mutation. An AHAS gene mutation that causes herbicide resistance may have negative pleiotropic effects on plant fitness. This study reports the effects of the Trp574Leu mutation on AHAS activity and reproductive traits of R. sativus. Eight of 17 feral radish accessions presented individuals resistant to metsulfuron-methyl at 0.5% to >90.0% and all the resistant individuals analyzed showed the Trp574Leu mutation. Without herbicide selection, the AHAS activity was 3.2-fold higher in the susceptible accession than in the resistant one. The resistant accession was >9000-fold more resistant to metsulfuron-methyl and imazethapyr than the susceptible accession. Under low intraspecific competition during two growing seasons, AHAS-resistant feral radish accessions showed 22-38% and 21-47% lower seed numbers and yield per plant than the susceptible accession. This is the first report of fitness cost associated with the AHAS Trp574Leu mutation in R. sativus populations. This fitness cost could reduce frequency of the resistant allele without herbicide selection. © 2018 Society of Chemical Industry.

Induction of the PDH bypass and upregulation of the ALDH7B4 in plants treated with herbicides inhibiting amino acid biosynthesis

Imazamox and glyphosate represent two classes of herbicides that inhibit the activity of acetohydroxyacid synthase in the branched-chain amino acid biosynthesis pathway and the activity of 5-enolpyruvylshikimate-3-phosphate synthase in the aromatic amino acid biosynthesis pathway, respectively. However, it is still unclear how imazamox and glyphosate lead to plant death. Both herbicides inhibit amino-acid biosynthesis and were found to induce ethanol fermentation in plants, but an Arabidopsis mutant deficient in alcohol dehydrogenase 1 was neither more susceptible nor more resistant than the wild-type to the herbicides. In this study, we investigated the effects of the amino acid biosynthesis inhibitors, imazamox and glyphosate, on the pyruvate dehydrogenase bypass reaction and fatty acid metabolism in A. thaliana. We found that the pyruvate dehydrogenase bypass was upregulated following the treatment by the two herbicides. Our results suggest that the Arabidopsis aldehyde dehydrogenase 7B4 gene might be participating in the pyruvate dehydrogenase bypass reaction. We evaluated the potential role of the aldehyde dehydrogenase 7B4 upon herbicide treatment in the plant defence mechanism. Plants that overexpressed the ALDH7B4 gene accumulated less soluble sugars, starch, and fatty acids and grew better than the wild-type after herbicide treatment. We discuss how the upregulation of the ALDH7B4 alleviates the effects of the herbicides, potentially through the detoxification of the metabolites produced in the pyruvate dehydrogenase bypass.

Development of imidazolinone herbicide tolerant borage (Borago officinalis L.)

Borage (Borago officinalis) is an annual herb that produces a high level of gamma-linolenic acid (GLA) in its seed oil. Due to the recognized health benefits of GLA, borage is now commercially cultivated worldwide. However, an herbicide-tolerant variety for effective weed management has not yet been developed. Here we report the generation and characterization of ethyl methanesulfonate (EMS) induced borage mutant lines tolerant to the herbicide imidazolinone. An EMS-mutagenized borage population was generated by using a series of concentrations of EMS to treat mature borage seeds. Screening of the M2 and M3 borage plants using an herbicide treatment resulted in the identification of two imidazolinone-tolerant lines. Sequence analysis of two acetohydroxyacid synthase (AHAS) genes, AHAS1 and AHAS2, from the mutant (tolerant) and wild type (susceptible) borage plants showed that single nucleotide substitutions which resulted in amino acid changes occurred in AHAS1 and AHAS2, respectively in the two tolerant lines. A KASP marker was then developed to differentiate the homozygous susceptible, homozygous tolerant and heterozygous borage plants. An in vitro assay showed that homozygous tolerant borage carrying the AHAS1 mutation retained significantly higher AHAS activity than susceptible borage across different imazamox concentrations. A herbicide dose response test indicated that the line with the AHAS1 mutation could tolerate four times the normally used field concentration of “Solo” herbicide.

The Role of a FAD Cofactor in the Regulation of Acetohydroxyacid Synthase by Redox Signaling Molecules

Acetohydroxyacid synthase (AHAS) catalyzes the first step of branched-chain amino acid (BCAA) biosynthesis, a pathway essential to the lifecycle of plants and microorganisms. This enzyme is of high interest because its inhibition is at the base of the exceptional potency of herbicides and potentially a target for the discovery of new antimicrobial drugs. The enzyme has conserved attributes from its predicted ancestor, pyruvate oxidase, such as a ubiquinone-binding site and the requirement for FAD as cofactor. Here, we show that these requirements are linked to the regulation of AHAS, in relationship to its anabolic function. Using various soluble quinone derivatives (e.g. ubiquinones), we reveal a new path of down-regulation of AHAS activity involving inhibition by oxidized redox-signaling molecules. The inhibition process relies on two factors specific to AHAS: (i) the requirement of a reduced FAD cofactor for the enzyme to be active and (ii) a characteristic slow rate of FAD reduction by the pyruvate oxidase side reaction of the enzyme. The mechanism of inhibition involves the oxidation of the FAD cofactor, leading to a time-dependent inhibition of AHAS correlated with the slow process of FAD re-reduction. The existence and conservation of such a complex mechanism suggests that the redox level of the environment regulates the BCAA biosynthesis pathway. This mode of regulation appears to be the foundation of the inhibitory activity of many of the commercial herbicides that target AHAS.

Cross-resistance pattern to four AHAS-inhibiting herbicides of tribenuron-methyl-resistant flixweed (Descurainia sophia) conferred by Asp-376-Glu mutation in AHAS

Flixweed (Descurainia sophia L.) is a problematic and widespread weed in winter wheat fields and has been controlled by tribenuron-methyl for more than twenty years in China. In this study, a flixweed accession (Hebei 25, HB25) with an Asp-376-Glu mutation in acetohydroxy acid synthase (AHAS) was identified and purified. The purified HB25 accession (pHB25) developed 758.1-fold resistance to tribenuron-methyl and exhibited obvious cross-resistance to four AHAS-inhibiting herbicides. The resistant/susceptible (R/S) ratios of 50% plant growth reduction (GR50) to herbicides of halosulfuron-methyl, flumetsulam, imazethapyr and pyribenzoxim were 346.1, 15.7, 8.1 and 7.1, respectively. The reduced AHAS sensitivities to four different AHAS-inhibiting herbicides, which were caused by the Asp-376-Glu mutation, were responsible for the resistance and cross-resistance to AHAS-inhibiting herbicides. The R/S ratios of 50% inhibition of AHAS activity (I50) to tribenuron-methyl, halosulfuron-methyl, flumetsulam, imazethapyr and pyribenzoxim were 844.5, 532.9, 74.5, 13.3 and 5.5, respectively. The results of AHAS activity in vitro were highly correlated with that of whole-plant response experiments.

Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium Thermotoga maritima has been purified and characterized (kcat ~100s−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.

Acetohydroxyacid synthase (AHAS) amino acid substitution Asp376Glu in Lolium perenne: effect on herbicide efficacy and plant growth

Recently, the acetohydroxyacid synthase (AHAS) amino acid substitution Asp376Glu was detected in a Lolium perenne population originating in France. This is the first documented occurrence of the Asp376Glu single-nucleotide polymorphism in a Lolium species. The effects on herbicide efficacy and plant fitness are presented. Separation of the original population into different genetic subgroups allowed the comparison of different L. perenne AHAS genotypes according to their susceptibility to AHAS-inhibiting herbicides. Root and shoot biomass accumulation as well as tiller production was analysed for resistant and sensitive populations. Genotype-specific AHAS activity and inhibition were studied in vitro, using recombinant Arabidopsis thaliana DNA. Plants expressing the AHAS Asp376Glu genotype were resistant against mesosulfuron + iodosulfuron, pyroxsulam and propoxycarbazone. To imazamox, reduced susceptibility for dosages below 140 g a.i. ha−1 was observed. In the absence of herbicide, no significant impact of the Asp376Glu genotype on shoot biomass and tiller accumulation could be observed, whereas root biomass of the Asp376Glu genotype was significantly reduced by 68 % compared to the AHAS genetic wild type. The AHAS enzyme study revealed reduced AHAS enzyme rate of 48 % for the Asp376Glu genotype compared to the AHAS genetic wild type. This study highlights the role of the amino acid exchange on resistance profile against the most relevant AHAS-inhibiting herbicides. First indications were found that fitness of the Asp376Glu genotype is hampered.

Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima

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±27U/mg and native molecular mass of 156±6kDa. 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±2mM and 246±7U/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.