Crystal structure of yeast acetohydroxyacid synthase: a target for herbicidal inhibitors

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis. The enzyme requires thiamin diphosphate and FAD for activity, but the latter is unexpected, because the reaction involves no oxidation or reduction. Due to its presence in plants, AHAS is a target for sulfonylurea and imidazolinone herbicides. Here, the crystal structure to 2.6 A resolution of the catalytic subunit of yeast AHAS is reported. The active site is located at the dimer interface and is near the proposed herbicide-binding site. The conformation of FAD and its position in the active site are defined. The structure of AHAS provides a starting point for the rational design of new herbicides.     

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.     

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.     

Relationships among the herbicide and functional sites of acetohydroxy acid synthase from Chlorella emersonii

The properties of acetohydroxy acid synthase (AHAS, EC 4.1.3.18) from wild-type Chlorella emersonii (var. Emersonii, CCAP-211/11n) and two spontaneous sulfometuron methyl (SMM)-resistant mutants were examined. The AHAS from both mutants was resistant to SMM and cross-resistant to imazapyr (IM) and the triazolopyrimidine sulfonanilide herbicide XRD-498 (TP). The more-SMM-resistant mutant had AHAS with altered catalytic parameters (K _m, specificity), but unchanged sensitivity to the feedback inhibitors valine and leucine. The second mutant enzyme was less sensitive to the feedback inhibitors, but had otherwise unchanged kinetic parameters. Inhibition-competition experiments indicated that the three herbicides (SMM, IM, TP) bind in a mutually exclusive manner, but that valine can bind simultaneously with SMM or TP. The three herbicide classes apparently bind to closely overlapping sites. We suggest that the results with C. emersonii and other organisms can all be explained if there are separate binding sites for herbicides, feedback inhibitors and substrates.Abbreviations AHAS acetohydroxy acid synthase - AL acetolactate - AHB acetohydroxybutyrate - IM imazapyr - TP triazolopyrimidine sulfonanilide herbicide XRD-498 - R enzyme specificity - SMM sulfometuron methyl This research was supported in part by the United States — Israel Binational Science Foundation (BSF), Jerusalem, Israel (Grant 86-00205) and the Fund for Basic Research, Israel Academy of Sciences.     

Computational study on the carboligation reaction of acetohidroxyacid synthase: New approach on the role of the HEThDP− intermediate

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate dependent enzyme that catalyses the decarboxylation of pyruvate to yield the hydroxyethyl‐thiamin diphosphate (ThDP) anion/enamine intermediate (HEThDP^–). This intermediate reacts with a second ketoacid to form acetolactate or acetohydroxybutyrate as products. Whereas the mechanism involved in the formation of HEThDP^– from pyruvate is well understood, the role of the enzyme in controlling the carboligation reaction of HEThDP^– has not been determined yet. In this work, molecular dynamics (MD) simulations were employed to identify the aminoacids involved in the carboligation stage. These MD studies were carried out over the catalytic subunit of yeast AHAS containing the reaction intermediate (HEThDP^–) and a second pyruvate molecule. Our results suggest that additional acid–base ionizable groups are not required to promote the catalytic cycle, in contrast with earlier proposals. This finding leads us to postulate that the formation of acetolactate relies on the acid–base properties of the HEThDP^– intermediate itself. PM3 semiempirical calculations were employed to obtain the energy profile of the proposed mechanism on a reduced model of the active site. These calculations confirm the role of HEThDP^– intermediate as the ionizable group that promotes the carboligation and product formation steps of the catalytic cycle. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Identification of the catalytic subunit of acetohydroxyacid synthase in Haemophilus influenzae and its potent inhibitors

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate- (ThDP)- and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids (BCAAs) leucine, isoleucine, and valine. The gene from Haemophilus influenzae that encodes the AHAS catalytic subunit was cloned, overexpressed in Escherichia coli BL21(DE3), and purified to homogeneity. The purified H. influenzae AHAS catalytic subunit (Hin-AHAS) appeared as a single band on SDS-PAGE gel, with a molecular mass of approximately 63 kDa. The enzyme catalyzes the condensation of two molecules of pyruvate to form acetolactate, with a K(m) of 9.2mM and the specific activity of 1.5 micromol/min/mg. The cofactor activation constant (K(c)=13.5 microM) and the dissociation constant (K(d)=3.3 microM) of ThDP were also determined by enzymatic assay and tryptophan fluorescence quenching studies, respectively. We screened a chemical library to discover new inhibitors of the Hin AHAS catalytic subunit. Through which, AVS-2087 (IC(50)=0.53 microM), KSW30191 (IC(50)=1.42 microM), and KHG20612 (IC(50)=4.91 microM) displayed potent inhibition as compare to sulfometuron methyl (IC(50)=276.31 microM).     

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.     

Role of a conserved arginine in the mechanism of acetohydroxyacid synthase: catalysis of condensation with a specific ketoacid substrate

The thiamin diphosphate (ThDP)-dependent bio-synthetic enzyme acetohydroxyacid synthase (AHAS) catalyzes decarboxylation of pyruvate and specific condensation of the resulting ThDP-bound two-carbon intermediate, hydroxyethyl-ThDP anion/enamine (HEThDP(-)), with a second ketoacid, to form acetolactate or acetohydroxybutyrate. Whereas the mechanism of formation of HEThDP(-) from pyruvate is well understood, the role of the enzyme in control of the carboligation reaction of HEThDP(-) is not. Recent crystal structures of yeast AHAS from Duggleby's laboratory suggested that an arginine residue might interact with the second ketoacid substrate. Mutagenesis of this completely conserved residue in Escherichia coli AHAS isozyme II (Arg(276)) confirms that it is required for rapid and specific reaction of the second ketoacid. In the mutant proteins, the normally rapid second phase of the reaction becomes rate-determining. A competing alternative nonnatural but stereospecific reaction of bound HEThDP(-) with benzaldehyde to form phenylacetylcarbinol (Engel, S., Vyazmensky, M., Geresh, S., Barak, Z., and Chipman, D. M. (2003) Biotechnol. Bioeng. 84, 833-840) provides a new tool for studying the fate of HEThDP(-) in AHAS, since the formation of the new product has a very different dependence on active site modifications than does acetohydroxyacid acid formation. The effects of mutagenesis of four different residues in the site on the rates and specificities of the normal and unnatural reactions support a critical role for Arg(276) in the stabilization of the transition states for ligation of the incoming second ketoacid with HEThDP(-) and/or for the breaking of the product-ThDP bond. This information makes it possible to engineer the active site so that it efficiently and preferentially catalyzes a new reaction.     

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.     

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.     

Probing the active center of benzaldehyde lyase with substitutions and the pseudosubstrate analogue benzoylphosphonic acid methyl ester

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of ( R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg (2+) as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 A (Protein Data Bank entry 3D7K ) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.     

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3μM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1μM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7? distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.     

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate-thiamin diphosphate adduct in AHAS I

Acetohydroxy acid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate (ThDP)-dependent decarboxylase-ligase that catalyzes the first common step in the biosynthesis of branched-chain amino acids. In the first stage of the reaction, pyruvate is decarboxylated and the reactive intermediate hydroxyethyl-ThDP carbanion/enamine is formed. In the second stage, the intermediate is ligated to another 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme I from Escherichia coli is unique among the AHAS isozymes in that it is not specific for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. It also appears to have a different mechanism for inhibition by valine than does AHAS III from E. coli. An investigation of this enzyme by directed mutagenesis and knowledge of detailed kinetics using the rapid mixing-quench NMR method or stopped-flow spectroscopy, as well as the use of alternative substrates, suggests that two residues determine most of the unique properties of AHAS I. Gln480 and Met476 in AHAS I replace the Trp and Leu residues conserved in other AHASs and lead to accelerated ligation and product release steps. This difference in kinetics accounts for the unique specificity, reversibility and allosteric response of AHAS I. The rate of decarboxylation of the initially formed 2-lactyl-ThDP intermediate is, in some AHAS I mutants, different for the alternative acceptors pyruvate and 2-KB, putting into question whether AHAS operates via a pure ping-pong mechanism. This finding might be compatible with a concerted mechanism (i.e. the formation of a ternary donor-acceptor:enzyme complex followed by covalent, ThDP-promoted catalysis with concerted decarboxylation-carboligation). It might alternatively be explained by an allosteric interaction between the multiple catalytic sites in AHAS.     

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.     

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.     

Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisiae

Transketolase from baker's yeast is a thiamin diphosphate-dependent enzyme in sugar metabolism that reconstitutes with various analogues of the coenzyme. The methylated analogues (4'-methylamino-thiamin diphosphate and N1'-methylated thiamin diphosphate) of the native cofactor were used to investigate the function of the aminopyrimidine moiety of the coenzyme in transketolase catalysis. For the wild-type transketolase complex with the 4'-methylamino analogue, no electron density was found for the methyl group in the X-ray structure, whereas in the complex with the N1'-methylated coenzyme the entire aminopyrimidine ring was disordered. This indicates a high flexibility of the respective parts of the enzyme-bound thiamin diphosphate analogues. In the E418A variant of transketolase reconstituted with N1'-methylated thiamin diphosphate, the electron density of the analogue was well defined and showed the typical V-conformation found in the wild-type holoenzyme [Lindqvist Y, Schneider G, Ermler U, Sundstrom M (1992) EMBO J11, 2373-2379]. The near-UV CD spectrum of the variant E418A reconstituted with N1'-methylated thiamin diphosphate was identical to that of the wild-type holoenzyme, while the CD spectrum of the variant combined with the unmodified cofactor did not overlap with that of the native protein. The activation of the analogues was measured by the H/D-exchange at C2. Methylation at the N1' position of the cofactor activated the enzyme-bound cofactor analogue (as shown by a fast H/D-exchange rate constant). The absorbance changes in the course of substrate turnover of the different complexes investigated (transient kinetics) revealed the stability of the alpha-carbanion/enamine as the key intermediate in cofactor action to be dependent on the functionality of the 4-aminopyrimidine moiety of thiamin diphosphate.     

Stable chloroplast transformation of the unicellular red alga Porphyridium species

Red algae are extremely attractive for biotechnology because they synthesize accessory photosynthetic pigments (phycobilins and carotenoids), unsaturated fatty acids, and unique cell wall sulfated polysaccharides. We report a high-efficiency chloroplast transformation system for the unicellular red microalga Porphyridium sp. This is the first genetic transformation system for Rhodophytes and is based on use of a mutant form of the gene encoding acetohydroxyacid synthase [AHAS(W492S)] as a dominant selectable marker. AHAS is the target enzyme of the herbicide sulfometuron methyl, which effectively inhibits growth of bacteria, fungi, plants, and algae. Biolistic transformation of synchronized Porphyridium sp. cells with the mutant AHAS(W492S) gene that confers herbicide resistance gave a high frequency of sulfomethuron methyl-resistant colonies. The mutant AHAS gene integrated into the chloroplast genome by homologous recombination. This system paves the way for expression of foreign genes in red algae and has important biotechnological implications.     

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.     

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.     

INTRASPECIFIC TRANSFER OF HERBICIDE RESISTANCE IN THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYCEAE) VIA PROTOPLAST FUSION

Stable progeny doubly resistant to the herbicides sulfometuron methyl (SMM) and diuron [3-(3,4-dichlorophenyl)-1,1-dimethylurea] (DCMU) were obtained at a frequency of 2% on fusion of protoplasts derived from mutants of Porphyridium sp. (UTEX 637) that were resistant only to SMM (strain SMR) or DCMU (strain DC-2). In the presence of both herbicides, only the fusion progeny could grow; both parental mutants were inhibited. In the absence of SMM, the activity of acetohydroxy acid synthase (AHAS) in the wild-type strain was similar to that in DC-2, exceeding that of SMR by up to 4.5-fold. AHAS activities of all fusion progeny were lower than those of the wild-type strain and DC-2 but higher than that of SMR. In the presence of SMM, AHAS activities of all tested fusion progeny ranged between those of the two parental mutants. This result indicates that both types of AHAS, the type resistant to SMM and the sensitive type, originating from SMR and DC-2, respectively, were expressed in the fusion progeny. In the presence of DCMU, the photosynthetic activity of SMR was completely inhibited, whereas that of DC-2 was unaffected. The photosynthetic activity of the fusion progeny in the presence of DCMU was slightly lower than that of DC-2. Both the cell volume and the DNA content of the fusion progeny were similar to those of the parents. However, the genetic nature of the fusion products has not yet been elucidated. To the best of our knowledge, this is the first report on transfer of herbicide resistance via protoplast fusion in algae.