Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium Thermotoga maritima has been purified and characterized (k_cat ~100 s^?1). It was found that the same enzyme also had the ability to catalyze the production of acetaldehyde and CO₂ from pyruvate, an activity of pyruvate decarboxylase (PDC) at a rate approximately 10% of its AHAS activity. Compared to the catalytic subunit, reconstitution of the individually expressed and purified catalytic and regulatory subunits of the AHAS stimulated both activities of PDC and AHAS. Both activities had similar pH and temperature profiles with an optimal pH of 7.0 and temperature of 85 °C. The enzyme kinetic parameters were determined, however, it showed a non-Michaelis-Menten kinetics for pyruvate only. This is the first report on the PDC activity of an AHAS and the second bifunctional enzyme that might be involved in the production of ethanol from pyruvate in hyperthermophilic microorganisms.     

Acetolactate Synthase Activity in Developing Maize (Zea mays L.) Kernels

Acetolactate synthase (EC 4.1.3.18) activity was examined in maize (Zea mays L.) endosperm and embryos as a function of kernel development. When assayed using unpurified homogenates, embryo acetolactate synthase activity appeared less sensitive to inhibition by leucine + valine and by the imidazolinone herbicide imazapyr than endosperm acetolactate synthase activity. Evidence is presented to show that pyruvate decarboxylase contributes to apparent acetolactate synthase activity in crude embryo extracts and a modification of the acetolactate synthase assay is proposed to correct for the presence of pyruvate decarboxylase in unpurified plant homogenates. Endosperm acetolactate synthase activity increased rapidly during early kernel development, reaching a maximum of 3 micromoles acetoin per hour per endosperm at 25 days after pollination. In contrast, embryo activity was low in young kernels and steadily increased throughout development to a maximum activity of 0.24 micromole per hour per embryo by 45 days after pollination. The sensitivity of both endosperm and embryo acetolactate synthase activities to feedback inhibition by leucine + valine did not change during kernel development. The results are compared to those found for other enzymes of nitrogen metabolism and discussed with respect to the potential roles of the embryo and endosperm in providing amino acids for storage protein synthesis.     

Properties of mutant acetolactate synthases resistant to triazolopyrimidine sulfonanilide

Triazolopyrimidine sulfanilides are a class of highly active herbicides whose primary target is acetolactate synthase. Spontaneous mutants of tobacco (Nicotiana tabacum) (KS-43) and cotton (Gossypium hirsutum) (PS-3 and DO-2) resistant to triazolopyrimidine sulfonanilide were selected in tissue culture. Acetolactate synthase partially purified from the three mutants were 80- to 1000-fold less sensitive to inhibition by the compound compared with the corresponding wild-type enzyme. The mutants also varied in the cross-resistance pattern to other acetolactate synthase inhibiting herbicides in the sulfonylurea, imidazolinone, and pyrimidyl-oxy-benzoate chemical families. Thus, acetolactate synthase from KS-43, PS-3, and DO-2 cultures have different mutations. The affinities for pyruvate, thiamine pyrophosphate, as well as the activity of the mutant enzymes were found to be comparable to the corresponding wild-type enzymes. However, the enzyme from PS-3 was highly resistant to feedback inhibition by valine and leucine. In contrast, acetolactate synthase from KS-43 and DO-2 were inhibited by valine and leucine to nearly the same extent as the wild-type enzymes. Also, PS-3 cultures accumulated much higher levels of the branched chain amino acids compared to the wild-type cotton culture. The mutation in the PS-3 enzyme has therefore rendered it insensitive to feedback regulation by valine and leucine.     

Engineering of carboligase activity reaction in Candida glabrata for acetoin production

Utilization of Candida glabrata overproducing pyruvate is a promising strategy for high-level acetoin production. Based on the known regulatory and metabolic information, acetaldehyde and thiamine were fed to identify the key nodes of carboligase activity reaction (CAR) pathway and provide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate decarboxylase, and butanediol dehydrogenase were selected to be manipulated for strengthening the CAR pathway. Following the rational metabolic engineering, the engineered strain exhibited increased acetoin biosynthesis (2.24 g/L). In addition, through in silico simulation and redox balance analysis, NADH was identified as the key factor restricting higher acetoin production. Correspondingly, after introduction of NADH oxidase, the final acetoin production was further increased to 7.33 g/L. By combining the rational metabolic engineering and cofactor engineering, the acetoin-producing C. glabrata was improved stepwise, opening a novel pathway for rational development of microorganisms for bioproduction.     

Enhancement of 1,3-propanediol production by expression of pyruvate decarboxylase and aldehyde dehydrogenase from Zymomonas mobilis in the acetolactate-synthase-deficient mutant of Klebsiella pneumoniae

The acetolactate synthase (als)-deficient mutant of Klebsiella pneumoniae fails to produce 1,3-propanediol (1,3-PD) or 2,3-butanediol (2,3-BD), and is defective in glycerol metabolism. In an effort to recover production of the industrially valuable 1,3-PD, we introduced the Zymomonas mobilis pyruvate decarboxylase (pdc) and aldehyde dehydrogenase (aldB) genes into the als-deficient mutant to activate the conversion of pyruvate to ethanol. Heterologous expression of pdc and aldB efficiently recovered glycerol metabolism in the 2,3-BD synthesis-defective mutant, enhancing the production of 1,3-PD by preventing the accumulation of pyruvate. Production of 1,3-PD in the pdc- and aldB-expressing als-deficient mutant was further enhanced by increasing the aeration rate. This system uses metabolic engineering to produce 1,3-PD while minimizing the generation of 2,3-BD, offering a breakthrough for the industrial production of 1,3-PD from crude glycerol.     

Structural Genes for a Newly Recognized Acetolactate Synthase in Escherichia coli K-12

Evidence is reported that shows the presence in Escherichia coli K-12 of a newly found acetolactate synthase. This enzyme is the product of two genes, ilvH and ilvI, both located very close to leu. Amber mutations have been found in both genes and therefore their products are polypeptides. Mutations in the ilvH gene cause the appearance of an acetolactate synthase activity which is relatively resistant to valine inhibition and can be separated by adsorption on hydroxylapatite from another activity present in the extract and more sensitive to valine inhibition than the former. A mutant altered in the ilvI gene was isolated among the revertants sensitive to valine inhibition of an ilvH mutant. Such a mutant lacks the resistant acetolactate synthase. A temperature-sensitive revertant of the ilvI mutant contained a temperature-sensitive acetolactate synthase. Thus ilvI is the structural gene for a specific acetolactate synthase. The activity of the ilvH gene product has been measured by adding an extract containing it to a purified ilvI acetolactate synthase, which, upon incubation, became more sensitive to valine inhibition. Conversely, a valine-sensitive acetolactate synthase (the product of the ilvH and the ilvI genes) became more resistant to valine inhibition upon incubation with an extract of a strain containing a missense ilvH gene product.     

Thiamine-dependent Accumulation of Tetramethylpyrazine Accompanying a Mutation in the Isoleucine-Valine Pathway

A mutant of Corynebacterium glutamicum was found to accumulate high concentrations of a material which crystallized upon cooling of the broth. The compound was identified as tetramethylpyrazine. The mutant was found to require isoleucine, valine, leucine, and pantothenate for growth. All four requirements probably result from the loss of a single enzyme of the isoleucine-valine pathway. Since similar mutants of Neurospora crassa accumulate acetoin, the present mutant probably forms tetramethylpyrazine from acetoin. Accumulation of tetramethylpyrazine was dependent upon addition of thiamine. This observation is consistent with the known activity of diphosphothiamine as a cofactor for the formation of acetolactate (a precursor of acetoin) from pyruvate.     

Purification and properties of a pyruvate carboligase from Zea mays cultured cells

An enzyme able to catalyze the synthesis of acetoin (3-hydroxy-2-butanon) from either pyruvate or acetaldehyde was isolated, partially purified and characterized from maize (Zea mays L. cv Black Mexican Sweet) cultured cells. It exhibited a maximal rate at neutral pH values, and strictly required thiamine pyrophosphate and a divalent cation for activity; on the contrary, unlike bacterial pyruvate oxidases, flavin was not required. Apparent Michaelis constants were 260±20 mM for pyruvate and 24±7 mM for acetaldehyde. Both substrate affinity and specificity were notably higher than those of pyruvate decarboxylase, an enzyme that also synthesizes acetoin as by-product. The partially purified protein was unable to catalyze the formation of other possible products of pyruvate decarboxylation, thus carboligase appears to be its main activity. Results suggest that acetoin synthesis may be of physiological significance in plants.     

Transient releases of acetaldehyde from tree leaves − products of a pyruvate overflow mechanism?

Emissions of acetaldehyde from tree leaves were investigated by proton‐transfer‐reaction mass spectrometry (PTR‐MS), a technique that allows simultaneous monitoring of different leaf volatiles, and confirmed by derivatization and high‐performance liquid chromatography analysis. Bursts of acetaldehyde were released by sycamore, aspen, cottonwood and maple leaves following light–dark transitions; isoprene emission served as a measure of chloroplastic processes. Acetaldehyde bursts were not accompanied by ethanol, but exposure of leaves to inhibitors of pyruvate transport or respiration, or anoxia, led to much larger releases of acetaldehyde, accompanied by ethanol under anoxic conditions. These same leaves have an oxidative pathway for ethanol present in the transpiration stream, resulting in acetaldehyde emissions that are inhibited in vivo by 4‐methylpyrazole, an alcohol dehydrogenase (Adh) inhibitor. Labelling of leaf volatiles with ¹³CO₂ suggested that the pools of cytosolic pyruvate, the proposed precursor of acetaldehyde bursts, were derived from both recent photosynthesis and cytosolic carbon sources. We hypothesize that releases of acetaldehyde during light–dark transitions result from a pyruvate overflow mechanism controlled by cytosolic pyruvate levels and pyruvate decarboxylase activity. These results suggest that leaves of woody plants contribute reactive acetaldehyde to the atmosphere under different conditions: (1) metabolic states that promote the accumulation of cytosolic pyruvate, triggering the pyruvate decarboxylase reaction; and (2) leaf ethanol oxidation resulting from ethanol transported from anoxic tissues.     

Flux through Citrate Synthase Limits the Growth of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain Escherichia coli KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD⁺. Expression of low-K_m Zymomonas mobilis pdc (pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In E. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of E. coli citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.     

Pyruvate decarboxylase is like acetolactate synthase (ILV2) and not like the pyruvate dehydrogenase E1 subunit

Protein sequences of pyruvate decarboxylase (PDC) derived from cloned yeast (Saccharomyces cerevisiae) and bacterial (Zymomonas mobilis) genes were compared with each other and with sequence databases. Extensive sequence similarities were found between them and with two others: cytochrome-linked pyruvate oxidase from Escherichia coli and acetolactate synthase (ilvI in E. coli; ILV2 gene in S. cerevisiae). All catalyse decarboxylation of pyruvate using thiamine pyrophosphate (TPP) as cofactor. General overall similarity suggests common ancestry for these enzymes. None of the sequences was similar to the E1 component of pyruvate dehydrogenase from E. coli which also decarboxylates pyruvate with the help of TPP.     

The phosphonopyruvate decarboxylase from Bacteroides fragilis

The Bacteroides fragilis capsular polysaccharide complex is the major virulence factor for abscess formation in human hosts. Polysaccharide B of this complex contains a 2-aminoethylphosphonate functional group. This functional group is synthesized in three steps, one of which is catalyzed by phosphonopyruvate decarboxylase. In this paper, we report the cloning and overexpression of the B. fragilis phosphonopyruvate decarboxylase gene (aepY), purification of the phosphonopyruvate decarboxylase recombinant protein, and the extensive characterization of the reaction that it catalyzes. The homotrimeric (41,184-Da subunit) phosphonopyruvate decarboxylase catalyzes (kcat = 10.2 +/- 0.3 s-1) the decarboxylation of phosphonopyruvate (Km = 3.2 +/- 0.2 microm) to phosphonoacetaldehyde (Ki = 15 +/- 2 microm) and carbon dioxide at an optimal pH range of 7.0-7.5. Thiamine pyrophosphate (Km = 13 +/- 2 microm) and certain divalent metal ions (Mg(II) Km = 82 +/- 8 microm; Mn(II) Km = 13 +/- 1 microm; Ca(II) Km = 78 +/- 6 microm) serve as cofactors. Phosphonopyruvate decarboxylase is a member of the alpha-ketodecarboxylase family that includes sulfopyruvate decarboxylase, acetohydroxy acid synthase/acetolactate synthase, benzoylformate decarboxylase, glyoxylate carboligase, indole pyruvate decarboxylase, pyruvate decarboxylase, the acetyl phosphate-producing pyruvate oxidase, and the acetate-producing pyruvate oxidase. The Mg(II) binding residue Asp-260, which is located within the thiamine pyrophosphate binding motif of the alpha-ketodecarboxylase family, was shown by site-directed mutagenesis to play an important role in catalysis. Pyruvate (kcat = 0.05 s-1, Km = 25 mm) and sulfopyruvate (kcat approximately 0.05 s-1; Ki = 200 +/- 20 microm) are slow substrates for the phosphonopyruvate decarboxylase, indicating that this enzyme is promiscuous.     

Catalytic acid-base groups in yeast pyruvate decarboxylase. 2. Insights into the specific roles of D28 and E477 from the rates and stereospecificity of formation of carboligase side products.

Yeast pyruvate decarboxylase (YPDC), in addition to forming its metabolic product acetaldehyde, can also carry out carboligase reactions in which the central enamine intermediate reacts with acetaldehyde or pyruvate (instead of the usual proton electrophile), resulting in the formation of acetoin and acetolactate, respectively (typically, 1% of the total reaction). Due to the common mechanism shared by the acetaldehyde-forming and carboligase reactions through decarboxylation, a detailed analysis of the rates and stereochemistry of the carboligase products formed by the E477Q, D28A, and D28N active center YPDC variants was undertaken. While substitution at either position led to an approximately 2-3 orders of magnitude lower catalytic efficiency in acetaldehyde formation, the rate of acetoin formation by the E477Q and D28N variants was higher than that by wild-type enzyme. Comparison of the steady-state data for acetaldehyde and acetoin formation revealed that the rate-limiting step for acetaldehyde formation by the D28A, H114F, H115F, and E477Q variants is a step post-decarboxylation. In contrast to the wild-type YPDC and the E477Q variant, the D28A and D28N variants could synthesize acetolactate as a major product. The lower overall rate of side-product formation by the D28A variant than wild-type enzyme attests to participation of D28 in steps leading up to and including decarboxylation. The results also provide insight into the state of ionization of the side chains examined. (R)-Acetoin is produced by the variants with greater enantiomeric excess than by wild-type YPDC. (S)-Acetolactate is the predominant enantiomer produced by the D28-substituted variants, the same configuration as produced by the related plant acetolactate synthase.     

Isolation,Purification, and Characterization of Acetolactate Synthase from a Lactococcus lactis Culture

Acetolactate synthase catalyzing the synthesis of -acetolactate was isolated from lactic acid bacteria Lactococcus lactissubsp. lactisbiovar. diacetylactis4 and purified. Acetolactate synthase was shown to be an allosteric enzyme with low affinity for the substrate: the K _Mfor pyruvate was 70 mM. The curve relating the dependence of enzyme activity to 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 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°C at pH 6.5. The molecular weight of acetolactate synthase was 150 kDa. 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.     

Kinetic mechanism of pyruvate decarboxylase. Evidence for a specific protonation of the enzymic intermediate.

Decarboxylation of pyruvate by pyruvate decarboxylase (EC 4.1.1.1) was performed in a reaction mixture containing 50% deuterium. The isolated product, acetaldehyde, was investigated directly by 1H NMR and by mass spectrometry after conversion to the 2,4-dinitrophenyl hydrazone. The protium content of 56% at acetaldehyde C1 demonstrates a specific protonation of the corresponding intermediate by the enzyme. Proton inventory studies and enzyme modification indicate the 4' amino group of the coenzyme, thiamine pyrophosphate, in an immonium structure being a possible proton donor. A 'partially concerted' mechanism is suggested for the reaction steps following the decarboxylation.     

Purification of porcine liver pyruvate dehydrogenase complex and characterization of its catalytic and regulatory properties.

Procedures are described for isolating highly purified porcine liver pyruvate and α-ketoglutarate dehydrogenase complexes. Rabbit serum stabilized these enzyme complexes in mitochondrial extracts, apparently by inhibiting lysosomal proteases. The complexes were purified by a three-step procedure involving fractionation with polyethylene glycol, pelleting through 12.5% sucrose, and a second fractionation under altered conditions with polyethylene glycol. Sedimentation equilibrium studies gave a molecular weight of 7.2 × 10⁶ for the liver pyruvate dehydrogenase complex. Kinetic parameters are presented for the reaction catalyzed by the pyruvate dehydrogenase complex and for the regulatory reactions catalyzed by the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. For the overall catalytic reaction, the competitive K_i to K_m ratio for NADH versus NAD⁺ and acetyl CoA versus CoA were 4.7 and 5.2, respectively. Near maximal stimulations of pyruvate dehydrogenase kinase by NADH and acetyl CoA were observed at NADH:NAD⁺ and acetyl CoA:CoA ratios of 0.15 and 0.5, respectively. The much lower ratios required for enhanced inactivation of the complex by pyruvate dehydrogenase kinase than for product inhibition indicate that the level of activity of the regulatory enzyme is not directly determined by the relative affinity of substrates and products of catalytic sites in the pyruvate dehydrogenase complex. In the pyruvate dehydrogenase kinase reaction, K⁺ and NH⁺₄ decreased the K_m for ATP and the competitive inhibition constants for ADP and (β,γ-methylene)adenosine triphosphate. Thiamine pyrophosphate strongly inhibited kinase activity. A high concentration of ADP did not alter the degree of inhibition by thiamine pyrophosphate nor did it increase the concentration of thiamine pyrophosphate required for half-maximal inhibition.     

New aspects on inhibition of plant acetolactate synthase by chlorsulfuron and imazaquin

The sulfonylurea herbicide chlorsulfuron and the imidazolinone herbicide imazaquin were shown to be noncompetitive and uncompetitive inhibitors, respectively, of purified acetolactate synthase from barley (Hordeum vulgare L.) with respect to pyruvate. From double-reciprocal plots of the time-dependent biphasic inhibition by chlorsulfuron, an initial apparent inhibition constant of 68 nanomolar was calculated (a 0 to 4 minute assay was used for the initial inhibition), and a final steady-state dissociation constant of 3 nanomolar was estimated. The corresponding constants for imazaquin were 10 and 0.55 micromolar. Specific binding of [¹⁴C]chlorsulfuron and [¹⁴C]imazaquin to purified acetolactate synthase from barley and partially purified enzyme from corn (Zea mays L.) could be demonstrated by gel filtration and equilibrium dialysis. Evidence is presented that the binding of the inhibitors to the enzyme follows the previously described mechanism of slow reversibility once excess inhibitor has been removed. However, after formation of the slowly reversible complex and subsequent dissociation, both chlorsulfuron and imazaquin seem to permanently inactivate acetolactate synthase. These results add a new feature to the mode of action of these herbicides with respect to their high herbicidal potency.     

Mutagenesis at asp27 of pyruvate decarboxylase from Zymomonas mobilis. Effect on its ability to form acetoin and acetolactate.

Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a bivalent cation as essential cofactors. The three-dimensional structure of PDC from Zymomonas mobilis (ZMPDC) shows that Asp27 (D27) is close to ThDP in the active site, and mutagenesis of this residue has suggested that it participates in catalysis. The normal product of the PDC reaction is acetaldehyde but it is known that the enzyme can also form acetoin as a by-product from the hydroxyethyl-ThDP reaction intermediate. This study focuses on the role of D27 in the production of acetoin and a second by-product, acetolactate. D27 in ZMPDC was altered to alanine (D27A) and this mutated protein, the wild-type, and two other previously constructed PDC mutants (D27E and D27N) were expressed and purified. Determination of the kinetic properties of D27A showed that the affinity of D27A for ThDP is decreased 30-fold, while the affinity for Mg2+ and the Michaelis constant for pyruvate were similar to those of the wild-type. The time-courses of their reactions were investigated. Each mutant has greatly reduced ability to produce acetaldehyde and acetoin compared with the wild-type PDC. However, the effect of these mutations on acetaldehyde production is greater than that on acetoin formation. The D27A mutant can also form acetolactate, whereas neither of the other mutants, nor the wild-type PDC, can do so. In addition, acetaldehyde formation and/or release are reversible in wild-type ZMPDC but irreversible for the mutants. The results are explained by a mechanism involving thermodynamic and geometric characteristics of the intermediates in the reaction.     

Comparative aspects of hydroxamic acid production by thiamine-dependent enzymes

The reactions of 4-chloronitrosobenzene with pyruvate decarboxylase and transketolase were investigated by use of a new high-pressure liquid chromatography method to determine any differences between these two enzymes with respect to hydroxamic acid production. In addition to the previously established difference in the type of hydroxamic acid produced by the two enzymes, several new and interesting differences in their reaction with nitrosoaromatics were discovered. Most notable was the finding that pyruvate decarboxylase gave 4-chlorophenylhydroxylamine as the major product from 4-chloronitrosobenzene, while transketolase did not produce any detectable hydroxylamine. A redox mechanism was proposed to account for arylhydroxylamine production by pyruvate decarboxylase. This redox mechanism can also explain hydroxamic acid production by pyruvate decarboxylase; however, a previously proposed nucleophilic reaction mechanism occurring simultaneously could not be totally disproven. Either of the two mechanisms is equally likely for transktolase action in view of the present evidence. Another major difference between these enzymes is that the rate of 4-chloronitrosobenzene conversion was found to be much faster for pyruvate decarboxylase than for transketolase when each enzyme was subjected to its own optimal reaction conditions. Transketolase displayed typical enzyme saturation kinetics with 4-chloronitrosobenzene with a K_m of 0.31 mM and V_max of 0.033 μmol ml^?1 min^?1 unit^?1 relative to 5 mMd-fructose 6-phosphate as sugar substrate. On the other hand, the reaction with pyruvate decarboxylase was first order in 4-chloronitrosobenzene with a combined rate constant of 2.0 min^?1 unit^?1 ml.