ABSTRACT
The fruit-like aroma of two valine-derived volatiles, isobutanol and isobutyl acetate, has great impact on the flavour and taste of alcoholic beverages, including sake, a traditional Japanese alcoholic beverage. With the growing worldwide interest in sake, breeding of yeast strains with intracellular valine accumulation is a promising approach to meet a demand for sakes with a variety of flavour and taste by increasing the valine-derived aromas. We here isolated a valine-accumulating sake yeast mutant (K7-V7) and identified a novel amino acid substitution, Ala31Thr, on Ilv6, a regulatory subunit for acetohydroxy acid synthase. Expression of the Ala31Thr variant Ilv6 conferred valine accumulation on the laboratory yeast cells, leading to increased isobutanol production. Additionally, enzymatic analysis revealed that Ala31Thr substitution in Ilv6 decreased sensitivity to feedback inhibition by valine. This study demonstrated for the first time that an N-terminal arm conserved in the regulatory subunit of fungal acetohydroxy acid synthase is involved in the allosteric regulation by valine. Moreover, sake brewed with strain K7-V7 contained 1.5-fold higher levels of isobutanol and isobutyl acetate than sake brewed with the parental strain. Our findings will contribute to the brewing of distinctive sakes and the development of yeast strains with increased production of valine-derived compounds.
Subject(s)
Acetolactate Synthase , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Acetolactate Synthase/genetics , Acetolactate Synthase/analysis , Acetolactate Synthase/metabolism , Alcoholic Beverages/microbiology , Valine/analysis , Valine/metabolismABSTRACT
Acetohydroxyacid synthase (AHAS) exists in plants and many microorganisms (including gut flora) but not in mammals, making it an attractive drug target. Fluorescent-based methods should be practical for high-throughput screening of inhibitors. Herein, we describe the development of the first AHAS fluorogenic assay based on an intramolecular charge transfer (ICT)-based fluorescent probe. The assay is facile, sensitive, and continuous and can be applied toward various AHASs from different species, AHAS mutants, and crude cell lysates. The fluorogenic assay was successfully applied for (1) high-throughput screening of commerical herbicides toward different AHASs for choosing matching herbicides, (2) identification of a Soybean AHAS gene with broad-spectrum herbicide resistance, and (3) identification of selective inhibitors toward intestinal-bacterial AHASs. Among the AHAS inhibitors, an active agent was found for selective inhibition of obesity-associated Ruminococcus torques growth, implying the possibility of AHAS inhibitors for the ultimate goal toward antiobesity therapeutics. The fluorogenic assay opens the door for high-throughput programs in AHAS-related fields, and the design principle might be applied for development of fluorogenic assays of other synthases.
Subject(s)
Acetolactate Synthase/analysis , Fluorescent Dyes/chemistry , Acetolactate Synthase/antagonists & inhibitors , Acetolactate Synthase/genetics , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Gastrointestinal Microbiome , Herbicide Resistance/genetics , High-Throughput Screening Assays , HumansABSTRACT
Acetohydroxy acid synthase (AHAS) and related enzymes catalyze the production of chiral compounds [(S)-acetolactate, (S)-acetohydroxybutyrate, or (R)-phenylacetylcarbinol] from achiral substrates (pyruvate, 2-ketobutyrate, or benzaldehyde). The common methods for the determination of AHAS activity have shortcomings. The colorimetric method for detection of acyloins formed from the products is tedious and does not allow time-resolved measurements. The continuous assay for consumption of pyruvate based on its absorbance at 333 nm, though convenient, is limited by the extremely small extinction coefficient of pyruvate, which results in a low signal-to-noise ratio and sensitivity to interfering absorbing compounds. Here, we report the use of circular dichroism spectroscopy for monitoring AHAS activity. This method, which exploits the optical activity of reaction products, displays a high signal-to-noise ratio and is easy to perform both in time-resolved and in commercial modes. In addition to AHAS, we examined the determination of activity of glyoxylate carboligase. This enzyme catalyzes the condensation of two molecules of glyoxylate to chiral tartronic acid semialdehyde. The use of circular dichroism also identifies the product of glyoxylate carboligase as being in the (R) configuration.
Subject(s)
Acetolactate Synthase/analysis , Acetolactate Synthase/antagonists & inhibitors , Acetolactate Synthase/metabolism , Carboxy-Lyases/metabolism , Circular Dichroism/methods , Escherichia coli/enzymology , Glyoxylates/pharmacology , Lactates/metabolism , Pyruvic Acid/metabolism , Stereoisomerism , Valine/pharmacologyABSTRACT
Ketol-acid reductoisomerase (EC 1.1.1.86) catalyzes the conversion of 2-aceto-2-hydroxyacids to 2-keto-3-hydroxyacids and their subsequent reduction by NADPH to 2,3-dihydroxyacids. The gene encoding the Escherichia coli enzyme was cloned and expressed as a hexahistidine-tagged fusion protein and the recombinant enzyme purified by metal-ligand affinity chromatography. The pure enzyme was tested for its ability to provide a sensitive and continuous coupled assay for acetohydroxyacid synthase (EC 4.1.3.18), the preceding enzyme in the pathway of branched-chain amino acid biosynthesis. An unexpected side reaction of ketol-acid reductoisomerase was observed in which it catalyzes the reduction of pyruvate. Although relatively slow, this side reaction is high enough to prohibit the use of this enzyme in a coupled assay for acetohydroxyacid synthase.
Subject(s)
Acetolactate Synthase/analysis , Alcohol Oxidoreductases/isolation & purification , Escherichia coli/enzymology , Acetolactate Synthase/metabolism , Alcohol Oxidoreductases/metabolism , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Indicators and Reagents , Ketol-Acid Reductoisomerase , Kinetics , Plasmids , Pyruvates/metabolism , Recombinant Fusion Proteins/isolation & purification , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
Examination of the ilvF locus at 54 min on the Escherichia coli K-12 chromosome revealed that it is a cryptic gene for expression of a valine-resistant acetohydroxy acid synthase (acetolactate synthase; EC 4.1.3.18) distinct from previously reported isozymes. A spontaneous mutation, ilvF663, yielded IlvF+ enzyme activity that was multivalently repressed by all three branched-chain amino acids, was completely insensitive to feedback inhibition, was highly stable at elevated temperatures, and expressed optimal activity at 50 degrees C. The IlvF+ enzyme activity was expressed in strains in which isozyme II was inactive because of the ilvG frameshift in the wild-type strain K-12 and isozymes I and III were inactivated by point mutations or deletions. Tn5 insertional mutagenesis yielded two IlvF- mutants, with the insertion in ilvF663 in each case. These observations suggest that the ilvF663 locus may be a coding region for a unique acetohydroxy acid synthase activity.
Subject(s)
Acetolactate Synthase/analysis , Escherichia coli/enzymology , Genes, Bacterial , Isoleucine/biosynthesis , Oxo-Acid-Lyases/analysis , Valine/biosynthesis , Acetolactate Synthase/genetics , Chromosome Mapping , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Hot Temperature , Hydrogen-Ion Concentration , Isoleucine/genetics , Molecular Weight , Mutation , Valine/geneticsABSTRACT
Acetohydroxy acid synthase (AHAS; EC 4.1.3.18) catalyzes the following two parallel, physiologically important reactions: condensation of two molecules of pyruvate to form acetolactate (AL), in the pathway to valine and leucine, and condensation of pyruvate plus 2-ketobutyrate to form acetohydroxybutyrate (AHB), in the pathway to isoleucine. We have determined the specificity ratio R with regard to these two reactions (where VAHB and VAL are rates of formation of the respective products) as follows: VAHB/VAL = R [2-ketobutyrate]/[pyruvate] for 14 enzymes from 10 procaryotic and eucaryotic organisms. Each organism considered has at least one AHAS of R greater than 20, and some appear to contain but a single biosynthetic AHAS. The implications of this for the design of the pathway are discussed. The selective pressure for high specificity for 2-ketobutyrate versus pyruvate implies that the 2-ketobutyrate concentration is much lower than the pyruvate concentration in all these organisms. It seems important for 2-ketobutyrate levels to be relatively low to avoid a variety of metabolic interferences. These results also reinforce the conclusion that biosynthetic AHAS isozymes of low R (1 to 2) are a special adaptation for heterotrophic growth on certain poor carbon sources. Two catabolic "pH 6 AL-synthesizing enzymes" are shown to be highly specific for AL formation only (R less than 0.1).
Subject(s)
Acetolactate Synthase/physiology , Oxo-Acid-Lyases/physiology , Acetolactate Synthase/analysis , Butyrates/metabolism , Hydrogen-Ion Concentration , Pyruvates/metabolism , Pyruvic Acid , Substrate SpecificityABSTRACT
Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, has received attention recently because of the finding that it is the site of action of several new herbicides. The most commonly used assay for detecting the enzyme is spectrophotometric involving an indirect detection of the product acetolactate. The assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex. There is considerable variability in the literature as to the details of this assay. We have investigated a number of factors involved in detecting AHAS in crude ammonium sulfate precipitates using this spectrophotometric method. Substrate and cofactor saturation levels, pH optimum, and temperature optimum have been determined. We have also optimized a number of factors involved in the generation and the detection of acetoin from acetolactate. The results of these experiments can serve as a reference for new investigators in the study of AHAS.
Subject(s)
Acetolactate Synthase/analysis , Oxo-Acid-Lyases/analysis , Acetoin/analysis , Hydrogen-Ion Concentration , Lactates/analysis , Plants/analysis , Substrate Specificity , TemperatureSubject(s)
Acetolactate Synthase/analysis , Escherichia coli/enzymology , Isoenzymes/analysis , Oxo-Acid-Lyases/analysis , Acetolactate Synthase/metabolism , Cell-Free System , Flavin-Adenine Dinucleotide , Hydrogen-Ion Concentration , Indicators and Reagents , Isoenzymes/metabolism , Spectrophotometry/methodsSubject(s)
Acetolactate Synthase/isolation & purification , Escherichia coli/enzymology , Isoenzymes/isolation & purification , Oxo-Acid-Lyases/isolation & purification , Acetolactate Synthase/analysis , Acetolactate Synthase/metabolism , Carbon Radioisotopes , Chromatography, Gel/methods , Chromatography, Ion Exchange/methods , Indicators and Reagents , Isoenzymes/analysis , Isoenzymes/metabolism , Kinetics , Macromolecular Substances , Molecular Weight , Radioisotope Dilution TechniqueSubject(s)
Acetolactate Synthase/isolation & purification , Isoenzymes/isolation & purification , Oxo-Acid-Lyases/isolation & purification , Salmonella typhimurium/isolation & purification , Acetolactate Synthase/analysis , Acetolactate Synthase/metabolism , Chromatography, Gel/methods , Chromatography, Ion Exchange/methods , Enzyme Stability , Indicators and Reagents , Isoenzymes/analysis , Isoenzymes/metabolism , Kinetics , Salmonella typhimurium/analysis , Salmonella typhimurium/metabolism , Spectrophotometry, Ultraviolet/methodsSubject(s)
Acetolactate Synthase/analysis , Escherichia coli/enzymology , Isoenzymes/analysis , Oxo-Acid-Lyases/analysis , Acetolactate Synthase/isolation & purification , Acetolactate Synthase/metabolism , Enzyme Stability , Indicators and Reagents , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Spectrophotometry, Ultraviolet/methodsABSTRACT
Acetohydroxyacid synthase I from Escherichia coli K-12 has been purified to near homogeneity. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two polypeptides, one with a molecular weight of 60,000 and one with a molecular weight of 9,500. These two polypeptides were present in constant proportion to each other and to enzyme activity. The molar ratio of the two polypeptides (Mr 9,500:60,000), estimated from stained polyacrylamide gels, was 1. Antisera raised against the 60,000 Mr polypeptide precipitated both the 60,000 and the 9,500 Mr polypeptides from extracts of cells labeled with [35S]methionine. The addition of sodium dodecyl sulfate before immunoprecipitation eliminated the smaller polypeptide, and only the larger one was recovered. The hydrodynamic properties of the native enzyme confirmed a previous report that the largest enzymatically active species has a molecular weight of about 200,000; this species contains both the 60,000- and 9,500-molecular-weight polypeptides.
Subject(s)
Acetolactate Synthase/isolation & purification , Escherichia coli/enzymology , Oxo-Acid-Lyases/isolation & purification , Acetolactate Synthase/analysis , Animals , Centrifugation, Zonal , Electrophoresis, Polyacrylamide Gel , Molecular Weight , Peptides/analysis , Peptides/isolation & purification , Precipitin Tests , RabbitsABSTRACT
Existence of one form of alpha-acetohydroxyacid synthetase shown by sucrose density gradient ultracentrifugation analysis, and inhibition in vitro by valine studies on the enzyme of Bacillus cereus T.