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In E. coli K‐12, the absence of unphosphorylated PtsN (unphospho‐PtsN) has been proposed to cause an L‐leucine‐sensitive growth phenotype (LeuS) by hyperactivated K+ uptake mediated impairment of the expression of the ilvBN operon, encoding subunits of the L‐valine (Val)‐sensitive acetohydroxyacid synthase I (AHAS I) that renders residual AHAS activity susceptible to inhibition by Leu and K+. This leads to AHAS insufficiency and a requirement for L‐isoleucine (Ile). Herein, we provide an alternate mechanism for the LeuS of the ∆ptsN mutant. Genetic and physiological studies with suppressors of the LeuS indicate that impaired expression of the ilvBN operon jointly caused by the absence of unphospho‐PtsN and the presence of Leu coupled to Leu‐mediated repression of expression of AHAS III leads to AHAS insufficiency rendering residual AHAS activity susceptible to chronic Val stress that may be generated by exogenous Leu. Hyperactivated K+ uptake and an elevated α‐ketobutyrate level mediate elevation of ilvBN expression and alleviate the LeuS. The requirement of unphospho‐PtsN as a positive regulator of ilvBN expression may buffer Ile biosynthesis against Leu‐mediated AHAS insufficiency and protect AHAS I function from chronic endogenous Val generated by Leu and could be realized in certain environments that impair AHAS function. [ABSTRACT FROM AUTHOR]

BACKGROUND Intensifying weed resistance has challenged the use of existing acetohydroxyacid synthase (AHAS)‐inhibiting herbicides. Hence, there is currently an urgent requirement for the discovery of a new AHAS inhibitor to effectively control AHAS herbicide‐resistant weed species produced by target mutation. RESULTS: To combat weed resistance caused by AHAS with P197L mutation, we built a structure library consisting of pyrimidinyl‐salicylic acid derivatives. Using the pharmacophore‐linked fragment virtual screening (PFVS) approach, hit compound 8 bearing 6‐phenoxymethyl substituent was identified as a potential AHAS inhibitor with antiresistance effect. Subsequently, derivatives of compound 8 were synthesized and evaluated for their inhibitory activities. The study of the enzyme‐based structure–activity relationship and structure−resistance relationship studies led to the discovery of a qualified candidate, 28. This compound not only significantly inhibited the activity of wild‐type Arabidopsis thaliana (At) AHAS and P197L mutant, but also exhibited good antiresistance properties (RF = 0.79). Notably, compared with bispyribac at 37.5–150 g of active ingredient per hectare (g a.i. ha–1), compound 27 exhibited higher growth inhibition against both sensitive and resistant Descurainia sophia, CONCLUSION: The title compounds have great potential to be developed as new leads to effectively control herbicide‐resistant weeds comprising AHAS with P197L mutation. Also, our study provided a positive case for discovering novel, potent and antiresistance inhibitors using a fragment‐based drug design approach. [ABSTRACT FROM AUTHOR]

BACKGROUND Acetohydroxyacid synthase large subunit 1 (Ahasl1) is a multiallelic locus involved in herbicide resistance in sunflower. Ahasl1‐1 and Ahasl1‐4 alleles harbor different point mutations that lead to different amino acid substitutions (Ala205Val and Trp574Leu, respectively). The objectives of this work were to evaluate the effect of these alleles at the enzymatic and whole‐plant levels, and to determine the dominance relationships for imazapyr and metsulfuron‐methyl herbicides. RESULTS: Resistant near‐isogenic lines showed significantly lower specific AHAS activity than susceptible near‐isoline. However, kinetic studies indicated that mutations did not change AHAS pyruvate affinity. Dose–response for six near‐isolines carrying different combinations of Ahasl1‐1 and Ahasl1‐4 alleles and two herbicides (imazapyr and metsulfuron‐methyl) were evaluated at whole‐plant and enzymatic levels. Ahasl1‐1 allele conferred moderate resistance to imazapyr and low resistance to metsulfuron‐methyl. Conversely, Ahasl1‐4 allele endowed high levels of resistance for both herbicides. Dominance of resistance at whole‐plant level showed a semi‐dominant behavior among the alleles for both herbicides. CONCLUSION: Ahasl1‐4 allele confers higher resistance levels than Ahasl1‐1 when evaluated with imazapyr and metsulfuron‐methyl. Dominance estimations suggested that both parental lines should carry a resistance trait when developing hybrids. © 2018 Society of Chemical Industry This study explores the effect of two sunflower Ahasl1 alleles (Ala205 and Trp574 mutations) at the enzymatic and whole‐plant levels, and the dominance relationships for imazapyr and metsulfuron‐methyl herbicides. [ABSTRACT FROM AUTHOR]

Summary: Sequence‐specific nucleases have been used to engineer targeted genome modifications in various plants. While targeted gene knockouts resulting in loss of function have been reported with relatively high rates of success, targeted gene editing using an exogenously supplied DNA repair template and site‐specific transgene integration has been more challenging. Here, we report the first application of zinc finger nuclease (ZFN)‐mediated, nonhomologous end‐joining (NHEJ)‐directed editing of a native gene in allohexaploid bread wheat to introduce, via a supplied DNA repair template, a specific single amino acid change into the coding sequence of acetohydroxyacid synthase (AHAS) to confer resistance to imidazolinone herbicides. We recovered edited wheat plants having the targeted amino acid modification in one or more AHAS homoalleles via direct selection for resistance to imazamox, an AHAS‐inhibiting imidazolinone herbicide. Using a cotransformation strategy based on chemical selection for an exogenous marker, we achieved a 1.2% recovery rate of edited plants having the desired amino acid change and a 2.9% recovery of plants with targeted mutations at the AHAS locus resulting in a loss‐of‐function gene knockout. The latter results demonstrate a broadly applicable approach to introduce targeted modifications into native genes for nonselectable traits. All ZFN‐mediated changes were faithfully transmitted to the next generation. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Descurainia sophia L., a problematic weed in winter wheat fields in China, has developed high resistance to tribenuron‐methyl. Amino acid substitutions at sites Pro197, Asp376 and Trp574 in the target acetohydroxyacid synthase (AHAS) were primarily responsible for D. sophia resistance to tribenuron‐methyl. In this study, purified subpopulations of D. sophia plants individually homozygous for a specific resistance mutation (Pro197Leu, Pro197His, Pro197Ser, Pro197Thr, Asp376Glu or Trp574Leu) in AHAS were generated, and the effects of resistance mutations on D. sophia resistance and AHAS characteristics were investigated. RESULTS: All resistance mutations in this study not only caused D. sophia to evolve 152‐ to 811‐fold resistance to tribenuron‐methyl but also greatly reduced AHAS sensitivity to tribenuron‐methyl and increased AHAS binding affinity for the substrate pyruvate, which was primarily responsible for D. sophia resistance. The molecular docking results indicated that these resistance mutations altered AHAS binding affinity for tribenuron‐methyl by reducing the hydrogen bonds and changing hydrophobic interactions. Compared with the wild‐type AHAS, these resistance mutations exhibited no significant effects on AHAS feedback inhibition by branched‐chain amino acids (BCAAs) at concentrations <0.08 m m. The altered AHAS sensitivity to feedback inhibition by BCAAs did not necessarily increase or decrease the free BCAAs in resistant D. sophia plants. CONCLUSION: The AHAS resistance mutations conferred D. sophia resistance to tribenuron‐methyl by decreasing the binding affinity for tribenuron‐methyl and/or increasing the binding affinity for pyruvate, but the mutations did not necessarily affect the biosynthesis of BCAAs in plants. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Summary: We investigated whether specific ALS‐inhibitor response marker‐genes (AIRMs) identified in the model dicotyledonous plant Arabidopsis thaliana (Brassicaceae) were conserved across phylogenetically very distant species. The most probable homologs of the 533 A. thalianaAIRMs were identified in the major monocotyledonous weeds Alopecurus myosuroides and Lolium sp. (Poaceae) using recently established transcriptome resources. Expression patterns of above 70% of the grass AIRMs homologs matched those of their A. thaliana counterparts. Pathways of response to acetolactate‐synthase (ALS)‐inhibiting herbicides seem therefore strongly conserved, even across very distant plant species. This opens new perspectives for unravelling the genetic determinants of non‐target‐site resistance (NTSR) to ALS inhibitors, especially those governing NTSR regulation that remains fully unknown to date. Model plants could be used in a first step to discover the genes involved in ALS‐inhibitor response pathways. Subsequent studies comparing sequence and expression of weed homologs of these genes should expedite the identification of candidates for the role of genes governing NTSR sensing and regulatory pathways. [ABSTRACT FROM AUTHOR]

Abstract: Acetohydroxyacid synthase (AHAS) is the first enzyme in the biosynthesis pathway of the branched‐chain amino acids, catalyzing the condensation of pyruvate with another molecule of pyruvate or with 2‐ketobutyrate, to produce 2‐acetolactate or 2‐acetohydroxybutyrate, respectively. The catalytic subunit of the dimeric enzyme has thiamin diphosphate (ThDP), a divalent metal ion, flavin adenine dinucleotide (FAD), and two molecules of oxygen (O2(I) and O2(II)) as cofactors. Here, crystal structures of Saccharomyces cerevisiae AHAS in complex with pyruvate provide novel insights into the mechanistic features of this enzyme including: i) The precise position taken by pyruvate molecules as they enter the active site (i. e. prior to catalysis occurring) with conformations suitable for the transfer of electrons to/from O2(I) and FAD; ii) The formation of ternary donor‐acceptor‐O2(I) complexes and iii) The location of O2(II) relative to the substrate showing that it plays a critical role in the organization of substrate for catalysis. These structural data, accompanied by electron paramagnetic resonance evidence that a radical is produced during AHAS catalysis, lead to the proposal that FAD and O2 are involved in an indirect one‐electron redox cycle. In this mechanism, the spatial configurations of O2 and FAD in the active site can allow electrons to be exchanged with the substrates and catalytic intermediates to satisfy and control the overall AHAS catalyzed reaction. [ABSTRACT FROM AUTHOR]

Acetohydroxyacid synthase (AHAS, ) is the first enzyme in the branched-chain amino acid biosynthesis pathway. Five of the most widely used commercial herbicides (i.e. sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl-benzoates and sulfonylamino-cabonyl-triazolinones) target this enzyme. Here we have determined the first crystal structure of a plant AHAS in the absence of any inhibitor (2.9 Å resolution) and it shows that the herbicide-binding site adopts a folded state even in the absence of an inhibitor. This is unexpected because the equivalent regions for herbicide binding in uninhibited Saccharomyces cerevisiae AHAS crystal structures are either disordered, or adopt a different fold when the herbicide is not present. In addition, the structure provides an explanation as to why some herbicides are more potent inhibitors of Arabidopsis thaliana AHAS compared to AHASs from other species (e.g. S. cerevisiae). The elucidation of the native structure of plant AHAS provides a new platform for future rational structure-based herbicide design efforts. Database The coordinates and structure factors for uninhibited AtAHAS have been deposited in the Protein Data Bank () with the PDB ID code . [ABSTRACT FROM AUTHOR]

BACKGOUND Acetohydroxyacid synthase ( AHAS; EC 2.2.1.6) is the first common enzyme in the biosynthetic pathway leading to the branched-chain amino acids in plants and a wide range of microorganisms. With the long-term and wide application of AHAS inhibitors, weed resistance is becoming a global problem, which leads to an urgent demand for novel inhibitors to antagonize both wild-type and resistant AHAS. RESULTS Pyrimidinyl salicylic acid derivatives, as one of the main classes of commercial AHAS herbicides, show potential anti-resistant bioactivity to wild-type and P197L mutant. In current work, a series of novel 2-benzoyloxy-6-pyrimidinyl salicylic acid derivatives were designed through fragment-based drug discovery. Fortunately, the newly synthesized compounds showed good inhibitory activity against both wild-type and P197L mutant. Some compounds not only had a lower resistance factor value but also showed excellent inhibitory activity against wild-type AHAS and P197L mutant. Furthermore, greenhouse experiments showed compound 11m displayed almost 100% inhibition against both wild-type and high-resistant Descurainia sophia at a dosage of 150 g a.i. ha−1. CONCLUSION The present work indicated that the 2-benzoyloxy-6-pyrimidinyl salicylic acid motif was well worth further optimization. Also, compound 11m could be used as a potential anti-resistant AHAS herbicide, which requires further research. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Sulfonylurea (SU) herbicides that target acetohydroxyacid synthase (AHAS, also called acetolactate synthase (ALS)) are regarded as one of the most important classes of herbicides, due to their extremely low toxicity towards mammals, ultra-low dosage application and high selectivity. However, mutations of AHAS to a herbicide-insensitive form appear to be a worldwide problem for acquired resistance. In this study, three mutated AHAS sequences were used. In the mutated AHASs, the proline (Pro) was replaced by one of threonine (Thr), alanine (Ala) or serine (Ser) in AHAS amino acid (AA) position 197Pro. To understand the herbicide resistance mechanism, we built dimeric AHAS complex models based on 1YBH of protein data bank (PDB) with SU herbicide, chlorimuron ethyl (CIE), using a homology. The three mutated AHASs in complex with CIE were analysed, and the resistance mechanism to SU herbicides was studied by molecular dynamics (MD) simulation performed using GROMACS 4.5.5. MD simulation analysis of wild-type and the three mutated dimeric AHAS-CIE complex systems revealed the conformational stability and changes in herbicide binding to dimeric AHAS system due to the mutation. Additionally, it showed that conformational change of amino acid residue 200Met (methionine) was associated with the imidazolinone herbicide resistance mechanism. According to our computation, the detailed mechanism of herbicide resistance was variable, depending on the type of mutated amino acid, providing new insights for designing herbicides specific to each mutant AHAS. [ABSTRACT FROM AUTHOR]

A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer's wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation. Transcriptional analysis of key genes involved in valine biosynthesis showed differences between the two strains that were consistent with the differences in wort diacetyl concentration. In particular, the ILV6 gene, encoding a regulatory subunit of acetohydroxy acid synthase, showed early transcription (only 6 h after inoculation) and up to five-fold greater expression in W34 compared to A153. This earlier transcription was observed for both orthologues of ILV6 in the S. pastorianus hybrid ( S. cerevisiae × S. eubayanus), although the S. cerevisiae form of ILV6 in W34 also showed a consistently higher transcript level throughout fermentation relative to the same gene in A153. Overexpression of either form of ILV6 (by placing it under the control of the PGK1 promoter) resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. The results confirm the role of the Ilv6 subunit in controlling α-acetolactate/diacetyl concentration and indicate no functional divergence between the two forms of Ilv6. The greater contribution of the S. cerevisiae ILV6 to acetolactate production in natural brewing yeast hybrids appears rather to be due to higher levels of transcription relative to the S. eubayanus form. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

BACKGROUND Sulfonylurea ( SU) herbicides are effective because they inhibit acetolactate synthase ( ALS), a key enzyme in branched-chain amino acid synthesis required for plant growth. A soybean line known as W4-4 was developed through rounds of seed mutagenesis and was demonstrated to have a high degree of ALS-based resistance to both post-emergence and pre-emergence applications of a variety of SU herbicides. This report describes the molecular and phenotypic characterization of the Als1 and Als2 mutations that confer herbicide resistance to SUs and other ALS inhibitors. RESULTS The mutations are shown to occur in two different ALS genes that reside on different chromosomes: Als1 ( P178S) on chromosome 4 and Als2 ( W560L) on chromosome 6 ( P197S and W574L in Arabidopsis thaliana). CONCLUSION Although the Als1 and Als2 genes are unlinked, the combination of these two mutations is synergistic for improved tolerance of soybeans to ALS-inhibiting herbicides. © 2014 DuPont Pioneer. Pest Management Science published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Acetohydroxyacid synthase ( AHAS) is a key target and has extensive application in the process of herbicide discovery. However, the problem of weed resistance is gradually serious with long-term excessive use of commercial AHAS-inhibiting herbicides, and so, it is urgent to develop novel herbicides. In this study, a virtual screening was performed based on the active site of AHAS. Then, the hit-10 compound with the IC50 value of 47.41 mg/L was selected as lead compound based on the biological testing. Subsequently, the optimization design and syntheses of lead compound were carried out according to the analyzing results of mechanism and receptor-/ligand-binding mode. Three novel 5-substituted benzyl-1,3,4-thiadiazol-2-carbamic acid phenyl ester derivatives were designed and synthesized. Bioactive assay results of the three target compounds showed that all of them displayed the higher inhibition than lead compound to AHAS, with the IC50 values in the 23.54-32.05 mg/L range, and the inhibition rates were increased by 30-50%. Finally, we also analyzed the possible inhibitory mechanism of the three target compounds based on the molecular docking between AHAS and target compounds. This study would provide a novel chemical structure for the discovery of novel AHAS herbicides. [ABSTRACT FROM AUTHOR]

Chlorsulfuron is the first commercialized sulfonylurea herbicide, which targets acetohydroxyacid synthase (AHAS). Mutations in AHAS have caused serious herbicide resistance to chlorsulfuron. Quantitative description of the herbicide resistance in molecular level will benefit the understanding of the resistance mechanism and aid the design of resistance-evading herbicide. We have recently established a MB-QSAR (Mutation-dependent Biomacromolecular Quantitative Structure-Activity Relationship) method to conduct the 3D-QSAR study in biomacromolecules. Herein, based on the herbicide resistance data measured for a series of AHAS mutants against chlorsulfuron, we constructed MB-QSAR models to quantitatively predict the herbicide resistance and interpret the structure resistance relationships for AHAS mutants against chlorsulfuron. Quite well correlations between the experimental and the predicted p Ki values were achieved for MB-QSAR/CoMFA ( q2=0.705, r2=0.918, r2pred=0.635) and MB-QSAR/CoMSIA ( q2=0.558, r2=0.940, r2pred=0.527) models, and interpretation of the MB-QSAR models gave chemical intuitive information to guide the resistance-evading herbicide design. [ABSTRACT FROM AUTHOR]

Parietochloris incisa is an oleaginous fresh water green microalga that accumulates an unusually high content of the valuable long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid within triacylglycerols in cytoplasmic lipid bodies. Here, we describe cloning and mutagenesis of the P. incisa acetohydroxyacid synthase ( PiAHAS) gene for use as an herbicide resistance selection marker for transformation. Use of an endogenous gene circumvents the risks and regulatory difficulties of cultivating antibiotic-resistant organisms. AHAS is present in plants and microorganisms where it catalyzes the first essential step in the synthesis of branched-chain amino acids. It is the target enzyme of the herbicide sulfometuron methyl (SMM), which effectively inhibits growth of bacteria and plants. Several point mutations of AHAS are known to confer herbicide resistance. We cloned the cDNA that encodes PiAHAS and introduced a W605S point mutation ( PimAHAS). Catalytic activity and herbicide resistance of the wild-type and mutant proteins were characterized in the AHAS-deficient E. coli, BUM1 strain. Cloned PiAHAS wild-type and mutant genes complemented AHAS-deficient bacterial growth. Furthermore, bacteria expressing the mutant PiAHAS exhibited high resistance to SMM. Purified PiAHAS wild-type and mutant proteins were assayed for enzymatic activity and herbicide resistance. The W605S mutation was shown to cause a twofold decrease in enzymatic activity and in affinity for the Pyruvate substrate. However, the mutant exhibited 7 orders of magnitude higher resistance to the SMM herbicide than that of the wild type. Biotechnol. Bioeng. 2012;109: 2340-2348. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

G oulart ICGR, M atzenbacher FO & M erotto A JR (2012). Differential germination pattern of rice cultivars resistant to imidazolinone herbicides carrying different acetolactate synthase gene mutations. Weed Research 52, 224-232. Summary Different mutations in the acetolactate synthase (ALS) gene can affect the germination pattern and facilitate the occurrence of ALS-inhibiting herbicide resistance in weedy red rice. The aim of this study was to evaluate the germination rates in imidazolinone-resistant rice cultivars carrying three different ALS gene mutations. Plant material consisted of the imidazolinone-resistant rice cultivars IRGA 422 CL, PUITÁ INTA CL and SATOR CL that carry the ALS mutations Gly654Glu, Ala122Thr and Ser653Asn, respectively, and the susceptible cultivar IRGA 417. Initially, the germination pattern of these cultivars was evaluated at temperatures of 15, 20, 25 and 30°C. Afterwards, five lots of each cultivar were evaluated at temperatures of 20 and 25°C to separate the environmental and genotypic effects. The environmental effect related to the different seed origin did not affect the germination rates of the rice cultivars. However, the germination pattern of the evaluated cultivars was different, mainly at 20°C. The time to reach 50% germination at 20°C for the imidazolinone-resistant rice cultivars IRGA 422 CL, SATOR CL and PUITÁ INTA CL was 6, 16 and 24 h earlier, respectively, than the susceptible cultivar IRGA 417. The PUITÁ INTA CL cultivar, which carries the mutation Ala122Thr, showed faster germination than the other herbicide-resistant and the susceptible cultivars at 20°C. The faster germination that resulted from the resistance to ALS-inhibiting herbicides could have different consequences for the establishment and competition of weedy red rice. [ABSTRACT FROM AUTHOR]

With 2 figures The development of imidazolinone (IMI) resistant sunflower cultivars represents a great advantage in controlling weeds. The aim of this study was to evaluate the response of sunflower genotypes to the presence of IMIs in a soil‐less bioassay. Six genotypes differing in IMI resistance were planted in pots filled with perlite and watered with different imazapyr doses. Differences among resistant, intermediate and susceptible genotypes were observed 8–15 days after planting. The susceptible genotype showed stunted plantlets. The intermediate genotypes developed reduced lateral roots when exposed to imazapyr. The resistant inbred line was only affected at the highest herbicide concentrations. Differences in aerial growth were also observed among genotypes. The bioassay described in this study is a rapid, simple and cost‐effective method for early screening herbicide resistant genotypes. This diagnostic test could be a useful tool when breeding for IMI resistance in sunflower. [ABSTRACT FROM AUTHOR]

The Ala205Val substitution in acetohydroxyacid synthase confers herbicide resistance in Solanum ptychanthum, but the effect on fitness is unknown. This study aimed to compare the germination, growth, seed production and competitiveness of resistant (R) and susceptible (S) populations under various light conditions in the field and in a controlled environment. Under optimal light conditions of high irradiance and high red/far-red, the S populations produced more seeds compared with R populations, despite no difference in germination and above-ground vegetative productivity. Any reduction in light intensity and red/far-red diminished the reproductive ability of both R and S populations. This effect was more pronounced for S, so that, under sub-optimal conditions, there were no differences between the two types. Replacement series with three ratios of R:S (16:0, 8:8 and 0:16) showed no competitive difference between the two types. Under optimal light conditions, the S populations always had a higher reproductive output and fitness than the R populations. This would likely cause S individuals to dominate in the absence of herbicide selection pressure, if light competition is reduced. [ABSTRACT FROM AUTHOR]

The role of thiamin diphosphate (ThDP) as a cofactor for enzymes has been known for many decades. This minireview covers the progress made in understanding the catalytic mechanism of ThDP-dependent enzymes through the use of ThDP analogues. Many such analogues have been synthesized and have provided information on the functional groups necessary for the binding and catalytic activity of the cofactor. Through these studies, the important role of hydrophobic interactions in stabilizing reaction intermediates in the catalytic cycle has been recognized. Stable analogues of intermediates in the ThDP-catalysed reaction mechanism have also been synthesized and crystallographic studies using these analogues have allowed enzyme structures to be solved that represent snapshots of the reaction in progress. As well as providing mechanistic information about ThDP-dependent enzymes, many analogues are potent inhibitors of these enzymes. The potential of these compounds as therapeutic targets and as important herbicidal agents is discussed. More recently, the way that ThDP regulates the genes for its own biosynthesis through the action of riboswitches has been discovered. This opens a new branch of thiamin research with the potential to provide new therapeutic targets in the fight against infection. [ABSTRACT FROM AUTHOR]

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is the first enzyme in the biosynthetic pathway of the branched-chain amino acids. It catalyzes the conversion of two molecules of pyruvate into 2-acetolactate or one molecule of pyruvate and one molecule of 2-ketobutyrate into 2-aceto-2-hydroxybutyrate. AHAS requires the cofactors thiamine diphosphate (ThDP), Mg2+ and FAD for activity. The herbicides that target this enzyme are effective in protecting a broad range of crops from weed species. However, resistance in the field is now a serious problem worldwide. To address this, two new sulfonylureas, monosulfuron and monosulfuron ester, have been developed as commercial herbicides in China. These molecules differ from the traditional sulfonylureas in that the heterocyclic ring attached to the nitrogen atom of the sulfonylurea bridge is monosubstituted rather than disubstituted. The structures of these compounds in complex with the catalytic subunit of Arabidopsis thaliana AHAS have been determined to 3.0 and 2.8 Å, respectively. In both complexes, these molecules are bound in the tunnel leading to the active site, such that the sole substituent of the heterocyclic ring is buried deepest and oriented towards the ThDP. Unlike the structures of Arabidopsis thaliana AHAS in complex with the classic disubstituted sulfonylureas, where ThDP is broken, this cofactor is intact and present most likely as the hydroxylethyl intermediate. [ABSTRACT FROM AUTHOR]

Crystal structures of two orthologs of the regulatory subunit of acetohydroxyacid synthase III (AHAS, EC 2.2.1.6) from Thermotoga maritima (TM0549) and Nitrosomonas europea (NE1324) were determined by single-wavelength anomalous diffraction methods with the use of selenomethionine derivatives at 2.3 Å and 2.5 Å, respectively. TM0549 and NE1324 share the same fold, and in both proteins the polypeptide chain contains two separate domains of a similar size. Each protein contains a C-terminal domain with ferredoxin-type fold and an N-terminal ACT domain, of which the latter is characteristic for several proteins involved in amino acid metabolism. The ferredoxin domain is stabilized by a calcium ion in the crystal structure of NE1324 and by a Mg(H2O)62+ ion in TM0549. Both TM0549 and NE1324 form dimeric assemblies in the crystal lattice. [ABSTRACT FROM AUTHOR]

Quantitative structure-activity relationship (QSAR) analysis has become one of the most effective approaches for optimizing lead compounds and designing new drugs. Although large number of quantum-chemical descriptors were defined and applied successfully, it is still a big challenge to develop a general quantum-chemical descriptor describing the bulk effects more directly and effectively. In this article, we defined a general quantum-chemical descriptor by characterizing the volume of electron cloud for specific substituent using the method of density functional theory. The application of our defined steric descriptors in the QSAR analysis of sulfonylurea analogues resulted in four QSAR models with high quality (the best model: q2 = 0.881, r2 = 0.901, n = 35, s = 0.401, F = 68.44), which indicated that this descriptor may provide an effective way for solving the problem how to directly describe steric effect in quantum chemistry-based QSAR studies. © 2006 Wiley Periodicals, Inc. J Comput Chem 27: 1571–1576, 2006 [ABSTRACT FROM AUTHOR]

Resistance to herbicides inhibiting acetolactate synthase (ALS) has been increasing at a faster rate than in any other herbicide group. The great majority of these cases are due to various single-nucleotide polymorphisms in the ALS gene endowing target site resistance. Many diagnostic techniques have been devised in order to confirm resistance and help producers to adopt the best management strategies. Recent advances in DNA technologies coupled with the knowledge of sequence information have allowed the development of accurate and rapid diagnostic tests. While whole plant-based diagnostic techniques such as seedling bioassays or enzyme-based in vitro bioassays provide accurate results, they tend to be labour- and/or space-intensive and will only respond to the particular herbicides tested, making resolution of cross-resistance patterns more difficult. Successful DNA-based diagnosis of ALS inhibitor resistance has been achieved with three main techniques, (1) restriction fragment length polymorphism, (2) polymerase chain reaction amplification of specific alleles and (3) denaturing high-performance liquid chromatography. All DNA-based techniques are relatively rapid and provide clear identification of the mutations causing resistance. Resistance based on non-target mechanisms is not identified by these DNA-based methods; however, given the prevalence of target site-based ALS inhibitor resistance, this is a minor inconvenience. [ABSTRACT FROM AUTHOR]

Abstract: Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate- (ThDP-) and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids such as leucine, isoleucine, and valine. The genes of AHAS from Mycobacterium tuberculosis were cloned, and overexpressed in E. coli and purified to homogeneity. The purified AHAS from M. tuberculosis is effectively inhibited by pyrazosulfuron ethyl (PSE), an inhibitor of plant AHAS enzyme, with the IC50 (inhibitory concentration 50%) of 0.87μM. The kinetic parameters of M. tuberculosis AHAS were determined, and an enzyme activity assay system using 96-well microplate was designed. After screening of a chemical library composed of 5600 compounds using the assay system, a new class of AHAS inhibitor was identified with the IC50 in the range of 1.8–2.6μM. One of the identified compounds (KHG20612) further showed growth inhibition activity against various strains of M. tuberculosis. The correlation of the inhibitory activity of the identified compound against AHAS to the cell growth inhibition activity suggested that AHAS might be served as a target protein for the development of novel anti-tuberculosis therapeutics. [Copyright &y& Elsevier]

RouxF, MatejicekA&ReboudX (2005) Response ofArabidopsis thalianato 22 ALS inhibitors: baseline toxicity and cross-resistance ofcsr1-1andcsr1-2resistant mutants.Weed Research45, 220–227.The baseline toxicity of 22 acetolactate synthase (ALS)-inhibiting herbicides and the cross-resistance patterns of chlorsulfuron- and imazapyr-resistant (R) lines on these 22 ALS-inhibiting herbicides were investigated using the model speciesArabidopsis thaliana. The 22 herbicides consisted of 18 sulfonylureas (SU), three imidazolinones (IMI) and one triazolopyrimidine (TP). The ED50 values (doses of herbicides required to reduce dry matter by 50%) of the post-emergence-treated Col and Ler susceptible (S) lines ranged from 22 to 4822 mg ha−1 and from 17 to 3143 mg ha−1 respectively. Thecsr1-1chlorsulfuron-resistant line (substitution of Pro197 to Ser) conferred a high resistance to the only TP tested as well as to nine SU herbicides (R:Sratio≥30), a low resistance to two SU herbicides (R:S≥5 and<30) and little or no resistance to the three IMI and seven other SU herbicides (R:S<5). This result contradicts the expectation that an ALS mutation selected by an SU herbicide confers high cross-resistance to other SU herbicides. We found that the efficacy of specific ALS inhibitors was different for different species and therefore could not be predicted from our results withA. thaliana; however, the cross-resistance patterns inA. thalianawere highly correlated with cross-resistance patterns in unrelated species with the same resistance mutation. These results have implications for resistance management. [ABSTRACT FROM AUTHOR]

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyses the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Mutants of the enzyme have been identified that are resistant to particular herbicides. However, the selectivity of these mutants towards various sulfonylureas and imidazolinones has not been determined systematically. Now that the structure of the yeast enzyme is known, both in the absence and presence of a bound herbicide, a detailed understanding of the molecular interactions between the enzyme and its inhibitors becomes possible. Here we construct 10 active mutants of yeast AHAS, purify the enzymes and determine their sensitivity to six sulfonylureas and three imidazolinones. An additional three active mutants were constructed with a view to increasing imidazolinone sensitivity. These three variants were purified and tested for their sensitivity to the imidazolinones only. Substantial differences are observed in the sensitivity of the 13 mutants to the various inhibitors and these differences are interpreted in terms of the structure of the herbicide-binding site on the enzyme. [ABSTRACT FROM AUTHOR]

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18; also known as acetolactate synthase), which catalyses the first reaction common to the biosynthesis of the branched-chain amino acids, L-valine, L-Ieucine and L-isoleucine, and is the target of several classes of herbicides, has been studied in hydroponically-grown seedlings of wheat (Triticum aestivum L. cv. Vulcan). Enzyme activity was greater in leaves than roots, reaching a maximum between 4 and 6 days after germination. AHAS was associated with the chloroplasts after centrifugation in a density gradient. A preparation of the enzyme was obtained from wheat leaves which gave a single band after electrophoresis in native gels but was resolved by denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis into three polypeptide bands of molecular mass 58, 57 and 15 kDa. The native molecular mass was approximately 128 kDa. AHAS had optimum activity at pH 7 and did not require the addition of flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP) and MgCl2 for activity. The enzyme did not display typical hyperbolic kinetics, in that the double reciprocal plot of activity against pyruvate concentration was non-linear. The concentration of pyruvate that gave half of the maximum activity was 4 mM. Sulfonylurea and imidazolinone herbicides were potent inhibitors of wheat leaf AHAS, with 50% inhibition being observed at concentrations of 0.6 and 0.3μM for chlorsulfuron and metsulfuron methyl, respectively, and at 2.5, 5 and 10 μM for imazaquin, imazethapyr and imazapyr. Inhibition by both classes of compounds was reversed by removal of the inhibitor. Progress curves of product formation against time in the presence of the herbicides were non-linear, and based on the assumption that inhibition by the sulfonylureas was of the slow, tight-binding type, estimates of 0.17 and 0.1 nM were obtained for the dissociation constants of chlorsulfuron and metsulfuron methyl, respectively, from the steady-state enzyme-inhibitor complex. [ABSTRACT FROM AUTHOR]

Acetohydroxyacid synthase (AHAS) is the key enzyme in the biosynthetic pathways of branched chain amino acids in bacteria. Since it does not exist in animal and plant cells, AHAS is an attractive target for developing antimicrobials and herbicides. In some bacteria, there is a single copy of AHAS, while in others there are multiple copies. Therefore, it is necessary to investigate the origin and evolutionary pathway of various AHASs in bacteria. In this study, all the available protein sequences of AHAS in bacteria were investigated, and an evolutionary model of AHAS in bacteria is proposed, according to gene structure, organization and phylogeny. Multiple copies of AHAS in some bacteria might be evolved from the single copy of AHAS, the ancestor. Gene duplication, domain deletion and horizontal gene transfer might occur during the evolution of this enzyme. The results show the biological significance of AHAS, help to understand the functions of various AHASs in bacteria, and would be useful for developing industrial production strains of branched chain amino acids or novel antimicrobials., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)