Preparation of a whole cell catalyst overexpressing acetohydroxyacid synthase of Thermotoga maritima and its application in the syntheses of α-hydroxyketones.

The large catalytic subunit of acetohydroxyacid synthase (AHAS, EC 2.2.1.6) of Thermotoga maritima (TmcAHAS) was prepared in this study. It possesses high specific activity and excellent stability. The protein and a whole cell catalyst overexpressing the protein were applied to the preparation of α-hydroxyketones including acetoin (AC), 3-hydroxy-2-pentanone (HP), and (R)-phenylacetylcarbinol (R-PAC). The results show that AC and HP could be produced in high yields (84% and 62%, respectively), while R-PAC could be synthesized in a high yield (about 78%) with an R/S ratio of 9:1. Therefore, TmcAHAS and the whole cell catalyst overexpressing the protein could be practically useful bio-catalysts in the preparation of α-hydroxyketones including AC, HP, and R-PAC. To the best of our knowledge, this is the first time that bacterial AHAS was used as a catalyst to prepare HP with a good yield, and also the first time that TmcAHAS was employed to synthesize AC and R-PAC.

Expression, purification and characterization of sll1981 protein from cyanobacterium Synechocystis sp. PCC6803.

The sll1981 protein from cyanobacterium Synechocystis sp. PCC6803 had been reported to exhibit acetolactate synthase (ALS) and L-myo-inositol-1-phosphate synthase (MIPS) activities previously. Based on amino acids sequences alignment, sll1981 protein was postulated to function as α-ketoglutarate decarboxylase (α-KGD), which played important role in completing cyanobacterial tricarboxylic acid (TCA) cycle. However the detailed enzymatic kinetics of sll1981 as ALS, MIPS and α-KGD were not determined yet. In this study, the recombinant sll1981 protein was purified from supernatant of E. coli cell and the substrate specificity of sll1981 towards pyruvate, d-glucose-6-phosphate and α-ketoglutarate was examined using homogenous recombinant sll1981. Steady-state kinetics results showed that sll1981 was a dual functional enzyme, which displayed much higher activity as α-KGD than as ALS. At the same time the MIPS activity of sll1981 was not detectable, although it was reported to be as MIPS previously. These findings not only confirmed the previous statement of the function of sll1981 as ALS and disputed the claimed function of sll1981 as MIPS, but also affirmed the new function of sll1981 as α-KGD. Therefore sll1981 was probably a key enzyme in completing the TCA cycle of Synechocystis sp. PCC6803.

Mutational analysis of critical residues of FAD-independent catabolic acetolactate synthase from Enterococcus faecalis V583.

Catabolic acetolactate synthase (cALS) from Enterococcus faecalis is a FAD-independent enzyme, which catalyzes the condensation of two molecules of pyruvate to produce acetolactate. Mutational and kinetic analyses of variants suggested the importance of H111, Q112, and Q411 residues for catalysis in cALS. The wild-type and variants were expressed as equally soluble proteins and co-migrated to a size of 60 kDa on SDS-PAGE. Importantly, H111 in cALS, which is widely present as phenylalanine in many other ThDP-dependent enzymes, plays a crucial role in substrate binding. Interestingly, the H111 variants, H111R and H111F, demonstrated altered specific activity of H111 variants with 17- and 26-fold increases in Km, respectively, compared to wild-type cALS. Furthermore, Q112 variants, Q112E, Q112N, and Q112V, exhibited significantly lower specific activity with 70-, 15-, and 10-fold higher Ks for ThDP, respectively. In the case of Q411, the variant Q411E showed a 10-fold rise in Km and a 20-fold increase in Ks for ThDP. Further, the molecular docking results indicated that the binding mode of ThDP was slightly affected in the variants of cALS. Based on these results, we suggest that H111 plays a role in substrate binding, and further suggest that Q112 and Q411 might be involved in ThDP binding of cALS.

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 1.7Å 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.

Expression, purification and preliminary crystallographic analysis of Rv3002c, the regulatory subunit of acetolactate synthase (IlvH) from Mycobacterium tuberculosis.

Branched amino-acid biosynthesis is important to bacterial pathogens such as Mycobacterium tuberculosis (Mtb), a microorganism that presently causes more deaths in humans than any other prokaryotic pathogen (http://www.who.int/tb). In this study, the molecular cloning, expression, purification, crystallization and preliminary crystallographic analysis of recombinant IlvH, the small regulatory subunit of acetohydroxylic acid synthase (AHAS) in Mtb, are reported. AHAS carries out the first common reaction in the biosynthesis of valine, leucine and isoleucine. AHAS is an essential enzyme in Mtb and its inactivation leads to a lethal phenotype [Sassetti et al. (2001), Proc. Natl Acad. Sci. USA, 98, 12712-12717]. Thus, inhibitors of AHAS could potentially be developed into novel anti-Mtb therapies.

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.

Safety assessment of a modified acetolactate synthase protein (GM-HRA) used as a selectable marker in genetically modified soybeans.

Acetolactate synthase (ALS) enzymes have been isolated from numerous organisms including soybeans (Glycine max; GM-ALS) and catalyze the first common step in biosynthesis of branched chain amino acids. Expression of an ALS protein (GM-HRA) with two amino acid changes relative to native GM-ALS protein in genetically modified soybeans confers tolerance to herbicidal active ingredients and can be used as a selectable transformation marker. The safety assessment of the GM-HRA protein is discussed. Bioinformatics comparison of the amino acid sequence did not identify similarities to known allergenic or toxic proteins. In vitro studies demonstrated rapid degradation in simulated gastric fluid (<30s) and intestinal fluid (<1min). The enzymatic activity was completely inactivated at 50 degrees C for 15 min demonstrating heat lability. The protein expressed in planta is not glycosylated and genetically modified soybeans expressing the GM-HRA protein produced similar protein/allergen profiles as its non-transgenic parental isoline. No adverse effects were observed in mice following acute oral exposure at a dose of at least 436 mg/kg of body weight or in a 28-day repeated dose dietary toxicity study at doses up to 1247 mg/kg of body weight/day. The results demonstrate GM-HRA protein safety when used in agricultural biotechnology.

Characterization of a mutation in the acetolactate synthase of Bacillus subtilis that causes a cold-sensitive phenotype.

The Bacillus subtilis laboratory strain JH642 shows a cold-sensitive phenotype after a temperature shift from 37 to 15 degrees C in comparison to wild type strain MR168. A mutation in the acetolactate synthase complex IlvBH was found to be partially responsible for this growth defect after cold shock. Via DNA sequencing, genetic and biochemical studies, this defect was characterized, which entails a substitution of two adenines to guanines in the ilvB gene. This results in an amino acid substitution from lysine at position 176 to glycine. As a consequence, the acetolactate synthase efficiency in strain JH642 was found to be reduced by 51-fold.

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

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.

Characterization of acetohydroxyacid synthase from Mycobacterium tuberculosis and the identification of its new inhibitor from the screening of a chemical library.

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 IC(50) (inhibitory concentration 50%) of 0.87 microM. 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 IC(50) in the range of 1.8-2.6 microM. 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.

Column flow reactor using acetohydroxyacid synthase I from Escherichia coli as catalyst in continuous synthesis of R-phenylacetyl carbinol.

We tested the possibility of utilizing acetohydroxyacid synthase I (AHAS I) from Escherichia coli in a continuous flow reactor for production of R-phenylacetyl carbinol (R-PAC). We constructed a fusion of the large, catalytic subunit of AHAS I with a cellulose binding domain (CBD). This allowed purification of the enzyme and its immobilization on cellulose in a single step. After immobilization, AHAS I is fully active and can be used as a catalyst in an R-PAC production unit, operating either in batch or continuous mode. We propose a simplified mechanistic model that can predict the product output of the AHAS I-catalyzed reaction. This model should be useful for optimization and scaling up of a R-PAC production unit, as demonstrated by a column flow reactor.

Characterization of two forms of acetolactate synthase from barley.

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target site for several classes of herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. Two forms of ALS (designated ALS I and ALS II) were separated from barley shoots by heparin affinity column chromatography. The molecular masses of native ALS I and ALS II were determined to be 248 kDa and 238 kDa by nondenaturing gel electrophoresis and activity staining. Similar molecular masses of two forms of ALS were confirmed by a Western blot analysis. SDS-PAGE and Western blot analysis showed that the molecular masses of the ALS I and ALS II subunits were identical--65 kDa. The two ALS forms exhibited different properties with respect to the values of K(m), pI and optimum pH, and sensitivity to inhibition by herbicides sulfonylurea and imidazolinone as well as to the feedback regulation by the end-product amino acids Val, Leu, and Ile. These results, therefore, suggest that the two ALS forms are not different polymeric forms of the same enzyme, but isozymes.

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.

Identification of the regulatory subunit of Arabidopsis thaliana acetohydroxyacid synthase and reconstitution with its catalytic subunit.

Acetohydroxyacid synthase (EC 4.1.3.18; AHAS) catalyzes the initial step in the formation of the branched-chain amino acids. The enzyme from most bacteria is composed of a catalytic subunit, and a smaller regulatory subunit that is required for full activity and for sensitivity to feedback regulation by valine. A similar arrangement was demonstrated recently for yeast AHAS, and a putative regulatory subunit of tobacco AHAS has also been reported. In this latter case, the enzyme reconstituted from its purified subunits remained insensitive to feedback inhibition, unlike the enzyme extracted from native plant sources. Here we have cloned, expressed in Escherichia coli, and purified the AHAS regulatory subunit of Arabidopsis thaliana. Combining the protein with the purified A. thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine, leucine, and isoleucine. Moreover, there is a strong synergy between the effects of leucine and valine, which closely mimics the properties of the native enzyme. The regulatory subunit contains a sequence repeat of approximately 180 residues, and we suggest that one repeat binds leucine while the second binds valine or isoleucine. This proposal is supported by reconstitution studies of the individual repeats, which were also cloned, expressed, and purified. The structure and properties of the regulatory subunit are reminiscent of the regulatory domain of threonine deaminase (EC 4.2.1.16), and it is suggested that the two proteins are evolutionarily related.

[Isolation, purification and properties of acetolactate synthase from cultured Lactococcus lactis]. / Vydelenie, ochistka i izuchenie svoistv atsetolaktatsintazy iz kul'tury Lactococcus lactis.

Acetolactate synthase catalyzing the synthesis of alpha-acetolactate was isolated from lactic acid bacteria Lactococcus lactis subsp. lactis biovar. diacetylactis 4 and purified. Acetolactate synthase was shown to be an allosteric enzyme with low affinity for the substrate: the Km for pyruvate was 70 mM. The curve relating the dependence of enzyme activity on pyruvate concentration had a sigmoid shape. The enzyme activity persisted for 24 h in the presence of stabilizers, pyruvate, and thiamine pyrophosphate. Acetolactate synthase had the pH optimums of 5.8 and 6.5-7.0 in acetate and phosphate buffers, respectively. The temperature optimum for this enzyme was 38-40 degrees C at pH 6.5. The molecular weight of acetolactate synthase was 150 kDa. In Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consisted of three identical subunits with a molecular weight of 55 kDa.

Mutagenesis studies on the sensitivity of Escherichia coli acetohydroxyacid synthase II to herbicides and valine.

Acetohydroxyacid synthase (EC 4.1.3.18, also known as acetolactate synthase) isoenzyme II from Escherichia coli is inhibited by sulphonylurea and imidazolinone herbicides, although it is much less sensitive than the plant enzyme. This isoenzyme is also unusual in that it is not inhibited by valine. Mutating S100 (Ser(100) in one-letter amino acid notation) of the catalytic subunit to proline increases its sensitivity to sulphonylureas, but not to imidazolinones. Mutating P536 to serine, as found in the plant enzyme, had little effect on the properties of the enzyme. Mutating E14 of the regulatory subunit to glycine, either alone or in combination with the H29N (His(29)-->Asn) change, did not affect valine-sensitivity.

Purification of acetohydroxy acid synthase by separation in an aqueous two-phase system.

Extraction in a polyethylene glycol (PEG)-phosphate aqueous two-phase system was considered as a primary step in purification of the acetohydroxy acid synthase III large catalytic subunit from an E. coli extract. Extraction optimization was achieved by varying the system parameters. Two systems with the following weight compositions were chosen for purification: PEG-2000 (16%)-phosphate (6%) and PEG-4000 (14%)-phosphate (5.5%)-KCl (8%), both at pH 7.0 and 1 mg total protein per 1 g system. Significant purification was achieved by a single extraction step with 70% recovery of the enzyme. After an additional ion-exchange chromatography step, pure enzyme was obtained in a 50% overall yield.

[Isolation and purification of acetolactate synthase and acetolactate decarboxylase from a Lactococcus lactis culture]. / Vydelenie i ochistka tsetolaktatsintazy i atsetolaktatdekaroksilazy iz kul'tury Lactococcus lactis.

Enzymes catalyzing the synthesis and subsequent transformation of alpha-acetolactate (AcL)--acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)--were isolated and partially purified from the cells of lactic acid bacteria Lactococcus lactis ssp. lactis biovar. diacetylactis strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358,300 U/mg protein (9% yield). The preparation of AcLDC, purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparent Km, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic, and the affinity of the enzyme for its substrate was high (Km = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.

Expression, purification, characterization, and reconstitution of the large and small subunits of yeast acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyzes the first step in the biosynthesis of the branched-chain amino acids. In bacteria, the enzyme has a large subunit containing the catalytic machinery and a small subunit with a regulatory role. In eucaryotes, the evidence for a regulatory subunit is largely indirect and circumstantial. We investigated the possibility that the yeast open reading frame YCL009c is an AHAS small subunit. Analysis of the DNA sequence shows that it contains all the appropriate transcription, translation and regulatory signals. YCL009c was shown to be expressed in yeast and the protein localized in mitochondria where it undergoes removal of a transit peptide targeting sequence. This putative small subunit protein (ilv6) and the catalytic subunit of yeast AHAS (ilv2) were each overexpressed in Escherichia coli and purified to near homogeneity. Reconstitution studies showed that the ilv6 protein stimulates the catalytic activity of the ilv2 protein by up to 7-fold (from 6.8 +/- 0.7 to 49.0 +/- 1.8 U/mg) and confers upon it sensitivity to inhibition by valine (Ki = 0.16 +/- 0.02 mM). Valine inhibition is partially reversed by ATP. The reconstitution is favored by high concentrations of potassium phosphate ( approximately 1 M) and at neutral pH. Under optimal conditions for reconstitution, a dissociation constant for the subunits of 70 +/- 7 nM was determined. Valine inhibition is partial, resulting in a specific activity that is similar to that of the ilv2 protein alone. However, measurements of the Km for substrate rule out the possibility that valine inhibition is accomplished by dissociation of the subunits.