Structural and functional evaluation of three well-conserved serine residues in tobacco acetohydroxyacid synthase

The first step in the common pathway for the biosynthesis of branched-chain amino acids (BCAAs) is catalyzed by acetohydroxyacid synthase (AHAS). The roles of three well-conserved serine residues (S167, S506, and S539) in tobacco AHAS were determined using site-directed mutagenesis. The mutations S167F and S506F were found to be inactive and abolished the binding affinity for cofactor FAD. The Far-UV CD spectrum of the inactive mutants was similar to that of wild-type enzyme, indicating no major conformational changes in the secondary structure. However, the active mutants, S167R, S506A, S506R, S539A, S539F and S539R, showed lower specific activities. Further, a homology model of tobacco AHAS was generated based on the crystal structure of yeast AHAS. In the model, the S167 and S506 residues were identified near the FAD binding site, while the S539 residue was found to near the ThDP binding site. The S539 mutants, S539A and S539R, showed strong resistance to three classes of herbicides, NC-311 (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine). In contrast, the active S167 and S506 mutants did not show any significant resistance to the herbicides, with the exception of S506R, which showed strong resistance to all herbicides. Thus, our results suggest that the S167 and S506 residues are essential for catalytic activity by playing a role in the FAD binding site. The S539 residue was found to be near the ThDP with an essential role in the catalytic activity and specific mutants of this residue (S539A and S539R) showed strong herbicide resistance as well.     

Elucidating the specificity of binding of sulfonylurea herbicides to acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) is the target for the sulfonylurea herbicides, which act as potent inhibitors of the enzyme. Chlorsulfuron (marketed as Glean) and sulfometuron methyl (marketed as Oust) are two commercially important members of this family of herbicides. Here we report crystal structures of yeast AHAS in complex with chlorsulfuron (at a resolution of 2.19 A), sulfometuron methyl (2.34 A), and two other sulfonylureas, metsulfuron methyl (2.29 A) and tribenuron methyl (2.58 A). The structures observed suggest why these inhibitors have different potencies and provide clues about the differential effects of mutations in the active site tunnel on various inhibitors. In all of the structures, the thiamin diphosphate cofactor is fragmented, possibly as the result of inhibitor binding. In addition to thiamin diphosphate, AHAS requires FAD for activity. Recently, it has been reported that reduction of FAD can occur as a minor side reaction due to reaction with the carbanion/enamine of the hydroxyethyl-ThDP intermediate that is formed midway through the catalytic cycle. Here we report that the isoalloxazine ring has a bent conformation that would account for its ability to accept electrons from the hydroxyethyl intermediate. Most sequence and mutation data suggest that yeast AHAS is a high-quality model for the plant enzyme.     

Roles of three well-conserved arginine residues in mediating the catalytic activity of tobacco acetohydroxy acid synthase

Acetohydroxy acid synthase (AHAS, EC 2.2.1.6; also known as acetolactate synthase, ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in plants and microorganisms. AHAS is the target of several classes of herbicides. In the present study, the role of three well-conserved arginine residues (R141, R372, and R376) in tobacco AHAS was determined by site-directed mutagenesis. The mutated enzymes, referred to as R141A, R141F, and R376F, were inactive and unable to bind to the cofactor, FAD. The inactive mutants had the same secondary structure as that of the wild type. The mutants R141K, R372F, and R376K exhibited much lower specific activities than the wild type, and moderate resistance to herbicides such as Londax, Cadre, and/or TP. The mutation R141K showed a strong reduction in activation efficiency by ThDP, while the mutations R372K and R376K showed a strong reductions in activation efficiency by FAD in comparison to the wild type enzyme. Taking into account the data presented here and the homology model constructed previously [Le et al. (2004) Biochem. Biophys. Res. Commun. 317, 930-938], it is suggested that the three amino acid residues studied (R141, R372, and R376) are located essentially at the enzyme active site, and, furthermore, that residues R372 and R376 are possibly responsible for the binding of the enzyme to FAD.     

Homology modeling of the structure of tobacco acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis

A reliable model of tobacco acetohydroxy acid synthase (AHAS) was obtained by homology modeling based on a yeast AHAS X-ray structure using the Swiss-Model server. Conserved residues at the dimer interface were identified, of which the functional roles of four residues, namely H142, E143, M489, and M542, were determined by site-directed mutagenesis. Eight mutants were successfully generated and purified, five of which (H142T, M489V, M542C, M542I, and M542V) were found to be inactive under various assay conditions. The H142K mutant was moderately altered in all kinetic parameters to a similar extent. In addition, the mutant was more thermo-labile than wild type enzyme. The E143A mutant increased the Km value more than 20-fold while other parameters were not significantly changed. All mutations carried out on residue M542 inactivated the enzyme. Though showing a single band on SDS-PAGE, the M542C mutant lost its native tertiary structure and was aggregated. Except M542C, each of the other mutants showed a secondary structure similar to that of wild type enzyme. Although all the inactive mutants were able to bind FAD, the mutants M489V and M542C showed a very low affinity for FAD. None of the active mutants constructed was strongly resistant to three tested herbicides. Taken together, the results suggest that the residues of H142, E143, M489, and M542 are essential for catalytic activity. Furthermore, it seems that H142 residue is involved in stabilizing the dimer interaction, while E143 residue may be involved in binding with substrate pyruvate. The data from the site-directed mutagenesis imply that the constructed homology model of tobacco AHAS is realistic.     

Molecular basis of sulfonylurea herbicide inhibition of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS) (acetolactate synthase, EC ) catalyzes the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides. These compounds are potent and selective inhibitors, but their binding site on AHAS has not been elucidated. Here we report the 2.8 A resolution crystal structure of yeast AHAS in complex with a sulfonylurea herbicide, chlorimuron ethyl. The inhibitor, which has a K(i) of 3.3 nm, blocks access to the active site and contacts multiple residues where mutation results in herbicide resistance. The structure provides a starting point for the rational design of further herbicidal compounds.     

Effect of Sucrose Concentration on the Infectivity of ϕX 174 DNA to Protoplast of Escherichia coli

The first step in branched-chain amino acid biosynthesis is catalyzed by acetohydroxyacid synthase (EC 2.2.1.6). This reaction involves decarboxylation of pyruvate followed by condensation with either an additional pyruvate molecule or with 2-oxobutyrate. The enzyme requires three cofactors, thiamine diphosphate (ThDP), a divalent ion, and flavin adenine dinucleotide (FAD). Escherichia coli contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the ilvB gene. In this study, the ilvB gene from E. coli K-12 was cloned into expression vector pETDuet-1, and was expressed in E. coli BL21 (DH3). The purified protein was identified on a 12% SDS–PAGE gel as a single band with a mass of 65 kDa. The optimum temperature, buffer, and pH for E. coli K-12 AHAS I were 37 °C, potassium phosphate buffer, and 7.5. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg⁺², ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 μM respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.     

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-A crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 A, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.     

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 TrpLeu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.     

Physical and kinetic properties of acetohydroxyacid synthase from wheat leaves

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-leucine 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 0.3 μM 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.     

Systematic characterization of mutations in yeast acetohydroxyacid synthase. Interpretation of herbicide-resistance data.

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.     

Characterization of acetohydroxyacid synthase I from Escherichia coli K-12 and identification of its inhibitors

The first step in branched-chain amino acid biosynthesis is catalyzed by acetohydroxyacid synthase (EC 2.2.1.6). This reaction involves decarboxylation of pyruvate followed by condensation with either an additional pyruvate molecule or with 2-oxobutyrate. The enzyme requires three cofactors, thiamine diphosphate (ThDP), a divalent ion, and flavin adenine dinucleotide (FAD). Escherichia coli contains three active isoenzymes, and acetohydroxyacid synthase I (AHAS I) large subunit is encoded by the ilvB gene. In this study, the ilvB gene from E. coli K-12 was cloned into expression vector pETDuet-1, and was expressed in E. coli BL21 (DH3). The purified protein was identified on a 12% SDS-PAGE gel as a single band with a mass of 65 kDa. The optimum temperature, buffer, and pH for E. coli K-12 AHAS I were 37 °C, potassium phosphate buffer, and 7.5. Km values for E. coli K-12 AHAS I binding to pyruvate, Mg(+2), ThDP, and FAD were 4.15, 1.26, 0.2 mM, and 0.61 μM respectively. Inhibition of purified AHAS I protein was determined with herbicides and new compounds.     

Mutations in corn (Zea mays L.) conferring resistance to imidazolinone herbicides

Summary Three corn (Zea mays L.) lines resistant to imidazolinone herbicides were developed by in vitro selection and plant regeneration. For all three lines, resistance is inherited as a single semidominant allele. The resistance alleles from resistant lines XA17, XI12, and QJ22 have been crossed into the inbred line B73, and in each case homozygotes are tolerant of commercial use rates of imidazolinone herbicides. All resistant selections have herbicide-resistant forms of acetohydroxyacid synthase (AHAS), the known site of action of imidazolinone herbicides. The herbicide-resistant phenotypes displayed at the whole plant level correlate directly with herbicide insensitivity of the AHAS activities of the selections. The AHAS activities from all three selections have normal feedback regulation by valine and leucine, and plants containing the mutations display a normal phenotype.     

Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids

Summary. The branched-chain amino acids are synthesized by plants, fungi and microorganisms, but not by animals. Therefore, the enzymes of this pathway are potential target sites for the development of antifungal agents, antimicrobials and herbicides. Most research has focused upon the first enzyme in this biosynthetic pathway, acetohydroxyacid synthase (AHAS) largely because it is the target site for many commercial herbicides. In this review we provide a brief overview of the important properties of each enzyme within the pathway and a detailed summary of the most recent AHAS research, against the perspective of work that has been carried out over the past 50 years.     

Biochemical evidence for multiple forms of acetohydroxy acid synthase in Spirulina platensis

Two isoforms of acetohydroxy acid synthase (AHAS), the first enzyme of the branched-chain amino acids biosynthetic pathway, were detected in cell-free extracts of the cyanobacterium Spirulina platensis and separated both by ion-exchange chromatography and by hydrophobic interaction. Several biochemical properties of the two putative isozymes were analysed and it was found that they differ for pH optimum, FAD requirement for both activity and stability, and for heat lability. The results were partially confirmed with the characterization of the enzyme extracted from a recombinant Escherichia coli strain transformed with one subcloned S. platensis coli strain transformed with one subcloned S. platensis AHAS gene. The approximate molecular mass of both AHAS activities, estimated by gel filtration, indicates that they are distinct isozymes and not different oligomeric species or aggregates of identical subunits.Abbreviations AHAS acetohydroxy acid synthase - DEAE cellulose diethylaminoethyl cellulose - DTT dithiothreitol - FAD flavin adenine dinucleotide - TPP thiamine pyrophosphate     

Multiple resistance to sulfonylureas and imidazolinones conferred by an acetohydroxyacid synthase gene with separate mutations for selective resistance

Summary The acetohydroxyacid synthase (AHAS) gene from the Arabidopsis thaliana mutant line GH90 carrying the imidazolinone resistance allele imr1 was cloned. Expression of the AHAS gene under the control of the CaMV 35S promoter in transgenic tobacco resulted in selective imidazolinone resistance, confirming that the single base-pair change found near the 3 end of the coding region of this gene is responsible for imidazolinone resistance. A chimeric AHAS gene containing both the imr1 mutation and the csr1 mutation, responsible for selective resistance to sulfonylurea herbicides, was constructed. It conferred on transgenic tobacco plants resistance to both sulfonylurea and imidazolinone herbicides. The data illustrate that a multiple-resistance phenotype can be achieved in an AHAS gene through combinations of separate mutations, each of which individually confers resistance to only one class of herbicides.     

Roles of lysine 219 and 255 residues in tobacco acetolactate synthase

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. The ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. The roles of three well-conserved lysine residues (K219, K255, K299) in tobacco ALS were determined using site-directed mutagenesis. The mutation of K219Q inactivated the enzyme and abolished the binding affinity for cofactor FAD. However, the secondary structure of the enzyme was not changed significantly by the mutation. Both mutants, K255F and K255Q, showed strong resistance to three classes of herbicides Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine). In addition, there was no difference in the secondary structures of wALS and K255F. On the other hand, the mutation of K299Q did not show any significant effect on the kinetic properties or any sensitivity to the herbicides. These results suggest that Lys219 is located at the active site and is likely involved in the binding of FAD, and that Lys255 is located at a binding site common for the three herbicides in tobacco ALS.     

大肠杆菌乙酰羟基酸合酶I调控亚基IlvN的结晶及其与配体缬氨酸的共结晶

乙酰羟基酸合酶(acetohydroxyacid synthase,AHAS)是生物体内支链氨基酸合成通路中的第一个通用酶,它是目前市售多种除草剂的靶标．AHAS通常由分子质量较大的催化亚基和分子质量较小的调控亚基组成．催化亚基结合催化必需的辅基(FAD、ThDP和Mg2+);调控亚基可以结合终产物(缬氨酸、亮氨酸或异亮氨酸)作为负反馈信号调节全酶的活性．大肠杆菌中AHAS有3个同工酶,每种同工酶都由催化亚基和调控亚基组成．大肠杆菌ilvN基因编码了AHAS同工酶Ⅰ的调控亚基．ilvN基因克隆到pET28a表达载体中,在大肠杆菌BL21(DE3)菌株中得到可溶性的大量表达．表达的蛋白质通过镍离子亲和层析和分子筛层析得到纯化．为了对调控亚基的调节机理有深入了解,对IlvN蛋白进行结晶并对蛋白质与其配体缬氨酸进行共结晶．IlvN蛋白晶体衍射能力为2.6 Å,IlvN与缬氨酸共结晶的晶体衍射能力为3.0 Å.     

Roles of conserved methionine residues in tobacco acetolactate synthase

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target of several classes of herbicides, including the sulfonylureas, the imidazolinones, and the triazolopyrimidines. The conserved methionine residues of ALS from plants were identified by multiple sequence alignment using ClustalW. The alignment of 17 ALS sequences from plants revealed 149 identical residues, seven of which were methionine residues. The roles of three well-conserved methionine residues (M350, M512, and M569) in tobacco ALS were determined using site-directed mutagenesis. The mutation of M350V, M512V, and M569V inactivated the enzyme and abolished the binding affinity for cofactor FAD. Nevertheless, the secondary structure of each of the mutants determined by CD spectrum was not affected significantly by the mutation. Both M350C and M569C mutants were strongly resistant to three classes of herbicides, Londax (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine), while M512C mutant did not show a significant resistance to the herbicides. The mutant M350C was more sensitive to pH change, while the mutant M569C showed a profile for pH dependence activity similar to that of wild type. These results suggest that M512 residue is likely located at or near the active site, and that M350 and M569 residues are probably located at the overlapping region between the active site and a common herbicide binding site.     

K252a, an indrocarbazole derivative, causes the membrane of myoblasts to enter a fusion-capable state

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in plants and microorganisms. ALS is the target of several structurally diverse classes of herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. The roles of three well-conserved histidine residues (H351, H392, and H487) in tobacco ALS were determined using site-directed mutagenesis. Both H487F and H487L mutations abolished the enzymatic activity as well as the binding affinity for the cofactor FAD. Nevertheless, the mutation of H487F did not affect the secondary structure of the ALS. The K(m) values of H351M, H351Q, and H351F are approximately 18-, 60-, and fivefold higher than that of the wild-type ALS, respectively. Moreover, the K(c) value of H351Q for FAD is about 137-fold higher than that of wALS. Mutants H351M and H351Q showed very strong resistance to Londax (a sulfonylurea) and Cadre (an imidazolinone), whereas mutant H351F was weakly resistant to them. However, the secondary structures of mutants H351M and H351Q appeared to be different from that of wALS. The mutation of H392M did not have any significant effect on the kinetic parameters nor the resistance to ALS-inhibiting herbicides. These results suggest that the His487 residue is located at the active site of the enzyme and is likely involved in the binding of cofactor FAD in tobacco ALS. Mutational analyses of the His351 residue imply that the active site of the ALS is probably close to its binding site of the herbicides, Londax and Cadre.     

Structure and mechanism of inhibition of plant acetohydroxyacid synthase

Plants and microorganisms synthesize valine, leucine and isoleucine via a common pathway in which the first reaction is catalysed by acetohydroxyacid synthase (AHAS, EC 2.2.1.6). This enzyme is of substantial importance because it is the target of several herbicides, including all members of the popular sulfonylurea and imidazolinone families. However, the emergence of resistant weeds due to mutations that interfere with the inhibition of AHAS is now a worldwide problem. Here we summarize recent ideas on the way in which these herbicides inhibit the enzyme, based on the 3D structure of Arabidopsis thaliana AHAS. This structure also reveals important clues for understanding how various mutations can lead to herbicide resistance.