Dihydroxyacid Dehydratase Research Articles

Linking central metabolism with increased pathway flux: L-valine accumulation by Corynebacterium glutamicum.

Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE, which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete L-valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since L-valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate. The same genes, ilvBNCD, overexpressed in an ilvA deletion mutant which is unable to synthesize L-isoleucine increased the concentration of this amino acid to 58 mM. A further dramatic increase was obtained when panBC was deleted, making the resulting mutant auxotrophic for D-pantothenate. When the resulting strain, C. glutamicum 13032DeltailvADeltapanBC with ilvBNCD overexpressed, was grown under limiting conditions it accumulated 91 mM L-valine. This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the L-valine biosynthesis pathway.

ChemInform Abstract: Synthesis of Modified Aldonic Acids and Studies of Their Substrate Efficiency for Dihydroxy Acid Dehydratase (DHAD).

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Escherichia coli Contains a Protein That Is Homologous in Function and N-terminal Sequence to the Protein Encoded by the nifS Gene of Azotobacter vinelandii and That Can Participate in the Synthesis of the Fe-S Cluster of Dihydroxy-acid Dehydratase

In this paper, I report the purification of a protein from Escherichia coli that is very similar in sequence, molecular weight, and the reactions it can catalyze to the protein encoded by the Azotobacter vinelandii nifS gene. This E. coli protein contains pyridoxal phosphate as a cofactor and catalyzes the removal of sulfur from cysteine to form alanine and S0. When dithiothreitol is present along with cysteine, the S0 formed is reduced to S2-. This protein has a reactive sulfhydryl group that is essential for activity. As isolated, this sulfhydryl group appears to be in a disulfide linkage with the sulfhydryl group from the phosphopantetheine moiety of the acyl carrier protein. The purified E. coli protein can mobilize the sulfur from cysteine and contribute it to the formation of a [4Fe-4S] cluster on the apoprotein of E. coli dihydroxy-acid dehydratase. A mechanism is proposed for the early stages of the synthesis of Fe-S clusters using this protein and sulfur in the S0 oxidation state.

Studies on the Synthesis of the Fe-S Cluster of Dihydroxy-acid Dehydratase in Escherichia coli Crude Extract: ISOLATION OF O-ACETYLSERINE SULFHYDRYLASES A AND B AND β-CYSTATHIONASE BASED ON THEIR ABILITY TO MOBILIZE SULFUR FROM CYSTEINE AND TO PARTICIPATE IN Fe-S CLUSTER SYNTHESIS

The apoprotein of Escherichia coli dihydroxy-acid dehydratase, which contains a catalytically essential [4Fe-4S] cluster in its active form, has been used as a substrate to investigate Fe-S cluster synthesis. The inactive apoprotein could be reactivated in vitro by factors present in the crude extract of E. coli and to a much smaller extent in the presence of Fe3+, S2-, and dithiothreitol. This reactivation occurs as a result of Fe-S cluster synthesis. It is anticipated that the Fe-S cluster synthesis observed in crude extracts in vitro may involve some of the components that participate in Fe-S cluster synthesis in vivo. The origin of the sulfur used to form Fe-S clusters was investigated. Four enzymatic activities in the crude extract of E. coli were found that can provide sulfur for Fe-S cluster synthesis in vitro by mobilizing the sulfur from cysteine. The purification of the proteins responsible for three of these activities is reported in this paper. The three proteins have been identified as O-acetylserine sulfhydrylase A, O-acetylserine sulfhydrylase B, and beta-cystathionase. The rate and extent of sulfide mobilization from cysteine in the reaction catalyzed by O-acetylserine sulfhydrylases A and B depend on the presence of nucleophiles that can add to the aminoacrylate formed on the enzyme following the removal of sulfide from cysteine. A new amino acid is formed when the nucleophiles add to the aminoacrylate. Sulfur mobilization by beta-cystathionase does not require a nucleophile, and the reaction is a minor variation on the cleavage of beta-cystathionine, with pyruvate, ammonia, and sulfide being the products. Once sulfur is mobilized by these enzymes, its efficient use in Fe-S cluster synthesis seems to be affected by the presence of yet unidentified factors present in crude extract. In crude extract and partially purified preparations from E. coli where these factors are present, the rapidity with which Fe-S clusters are formed and the efficiency with which sulfur is used imply an orderly controlled formation of Fe-S clusters that is generally typified by enzymatic reactions.

Synthesis of Modified Aldonic Acids and Studies of Their Substrate Efficiency for Dihydroxy Acid Dehydratase (DHAD)

Modified aldopentonic and aldohexonic acids were synthesized in order to study the electronic requirements for a successful enzymatic conversion into their corresponding 2-keto 3-deoxy analogues by dihydroxy acid dehydratase (DHAD), an enzyme from the biosynthetic pathway of branched chain amino acids. Analytical tests with the novel artificial substrates (18)-(21) and (27) provided evidence that the amount of conversion could be enhanced by replacement of the hydroxy group at C4 of L-arabinonic acid (21) with less electron-withdrawing, ambivalent or electron-donating substituents. Modified aldohexonic acids were no substrates for DHAD, perhaps due to less perfect binding to the active site presumably for steric reasons. For 4-deoxy-L-threo-pentonic acid (18) the enzymatic conversion into 3,4-dideoxy-2-ketopentonic acid (29) by DHAD could be achieved on a preparative scale.

A Metabolic Enzyme That Rapidly Produces Superoxide, Fumarate Reductase of Escherichia coli

Aerobic organisms synthesize superoxide dismutases in order to escape injury from endogenous superoxide. An earlier study of Escherichia coli indicated that intracellular superoxide is formed primarily by autoxidation of components of the respiratory chain. In order to identify those components, inverted respiratory vesicles were incubated with five respiratory substrates. In most cases, essentially all of the superoxide was formed through autoxidation of fumarate reductase, despite the paucity of this anaerobic terminal oxidase in the aerobic cells from which the vesicles were prepared. In contrast, most dehydrogenases, the respiratory quinones, and the cytochrome oxidases did not produce any detectable superoxide. The propensity of fumarate reductase to generate superoxide could conceivably deluge cells with superoxide when anaerobic cells, which contain abundant fumarate reductase, enter an aerobic habitat. In fact, deletion or overexpression of the frd structural genes improved and retarded, respectively, the outgrowth of superoxide dismutase-attenuated cells when they were abruptly aerated, suggesting that fumarate reductase is a major source of superoxide in vivo. Steric inhibitors that bind adjacent to the flavin completely blocked superoxide production, indicating that the flavin, rather than an iron-sulfur cluster, is the direct electron donor to oxygen. Since the turnover numbers for superoxide formation by other flavoenzymes are orders of magnitude lower than that of fumarate reductase (1600 min-1), additional steric or electronic factors must accelerate its autoxidation.

The role and properties of teh [Fe2S2] centers in Escherichia coli biotin synthase and spinach dihydroxy acid dehydratase

The role and properties of teh [Fe2S2] centers in Escherichia coli biotin synthase and spinach dihydroxy acid dehydratase

Dihydroxy-acid Dehydratase, a (4Fe-4S) Cluster-Containing Enzyme in Escherichia coli: Effects of Intracellular Superoxide Dismutase on Its Inactivation by Oxidant Stress

Dihydroxy-acid dehydratase (DHAD) has a [4Fe-4S] cluster and is reported to be facilely inactivated by oxidant stress, To directly assess the biological effects in vivo of superoxide dismutase (SOD) on the oxidant sensitivity of DHAD, we used an Escherichia coli K-12 parent strain (CGSC5073) and derived strains OB 1, OB 2, and OB 3 that lacked one of or both FeSOD and MnSOD, In the K-12 parent strain half the cellular DHAD activity was lost in 15 min at 0.8 atm oxygen, less than 10 μM aerobic nitrofurantoin, or about 5 μM aerobic paraquat (PQ) and in about 1 min at 10 μM aerobic PQ. Oxygen and metabolism were required for PQ to inactivate DHAD in cells; adding dithiothreitol to cell-free extracts did not restore DHAD activity. The Km was not appreciably changed for DHAD that was 50 and 70% inactivated in cells, respectively, by hyperbaric oxygen (HBO) and PQ, compared to cells in exponential, aerobic growth, Thus, active site oxidative impairment of individual enzyme molecules apparently was all-or-none. DHAD activity was greatly decreased when measured in extracts made from strains that lacked both SODs unless SOD was added to cell suspensions before extracts were made, DHAD was more sensitive in strains lacking both SODs than in the parent strain to inactivation by aerobic PQ and HBO, Anaerobic (compared to aerobic) growth increased DHAD specific activity by 20% or less in the parent strain and in strains OB 1 and OB 2 (lacking MnSOD and FeSOD, respectively); however, in strain OB 3 (lacking both SODs) DHAD was increased 60%. DHAD was partially inactivated by the oxidant stress of aerobic growth, but remained in a form detectable by DHAD antibody, and the ratio of active to inactive DHAD decreased greatly in cells lacking SOD. Thus, SOD helped maintain DHAD as an active holoenzyme and benefitted cells growing aerobically or when exposed to low levels of PQ.

Conversion of aldonic acids to their corresponding 2-keto-3-deoxy-analogs by the non-carbohydrate enzyme dihydroxy acid dehydratase (DHAD)

Aldonic acids containing four to six carbon atoms were tested as potential substrates of dihydroxy acid dehydratase (DHAD), an enzyme from the biosynthetic pathway of branched chain amino acids. Novel assay systems for observing the course of DHAD catalysed reactions were developed in order to adapt the enzyme to extended practical applications. Kinetic studies for the new substrates ( 12 13 ) as well as inhibitor kinetics for the substrate analogue 3-deoxy-aldonic acids ( 25, 27 and 29) were performed. These gave indications for the restrictions of substrate modifications and contributed to the understanding of the individual effects. Finally l-threonic acid ( 12) and d-erythronic acid ( 13) could be successfully applied as substrates for DHAD and this led to the chemoenzymatic synthesis of their 2-keto-3-deoxy-analogue ( 20) in a preparative scale.

Studies on the Active Site of Dihydroxy-acid Dehydratase

Several classes of substrate analogs of dihydroxy-acid dehydratase have been tested as inhibitors of this enzyme in an attempt to characterize its binding site and find what modifications in substrate structure lead to an affinity higher than that of the natural substrates. The substrate analogs were tested on dihydroxy-acid dehydratase from both spinach and Escherichia coli. One modification of the substrate that led to as much as a 1000-fold increase in binding affinity was replacement of the 3-hydroxyl group with a thiol. It has been shown previously that the 3-hydroxyl group of the substrate becomes a ligand for one Fe of the Fe-S clusters of these enzymes on binding to their active sites. It seems likely then that the tighter binding of the thiol containing analogs is due to the thiol group becoming a ligand to an iron of the Fe-S clusters of these enzymes. A second modification in substrate that led to as much as 1000-fold increase in binding affinity was the addition of a large lipophilic group. This suggests there is a large hydrophobic pocket or hydrophobic surface near the active site of dihydroxy-acid dehydratase. A modification in substrate that led to as much as a 50-fold increase in binding was the replacement of the carboxyl group of the substrate with phosphonate; however, this increase was limited to substrate analogs without a polar functionality on the carbon β to the phosphonate group. Bromopyruvate was found to irreversibly inactivate dihydroxy-acid dehydratase. Each good inhibitor we found was active on spinach dihydroxy-acid dehydratase and E. coli dihydroxy-acid dehydratase to a similar extent suggesting the active sites of the enzymes from these two organisms are similar. Some of the better inhibitors described in this report have mild herbicidal activity.

The inactivation of dihydroxy-acid dehydratase in Escherichia coli treated with hyperbaric oxygen occurs because of the destruction of its Fe-S cluster, but the enzyme remains in the cell in a form that can be reactivated.

The enzyme dihydroxy-acid dehydratase previously has been shown to be inactivated in vivo in Escherichia coli within minutes of exposure to hyperbaric O2. In this paper, we show its inactivation is due to the destruction of its catalytically active [4Fe-4S] cluster. The inactivation is not followed by an appreciable decrease in the amount of dihydroxy-acid dehydratase protein as determined by Western blots. Thus, the protein from the inactivated enzyme remains unproteolyzed in the cells. Dihydroxy-acid dehydratase activity recovers after the cells treated with hyperbaric O2 are returned to ambient oxygen. Since this recovery in activity is not accompanied by a significant increase in dihydroxy-acid dehydratase protein and is not prevented by chloramphenicol, it appears primarily to be due to reactivation of the previously inactivated enzyme. The reactivation occurs by reconstitution of the enzyme's Fe-S cluster. These results demonstrate that this enzyme can cycle between forms in which the Fe-S cluster is either present or absent. The facile ability to cycle between these two forms would be compatible with a regulatory role in addition to a catalytic role for this enzyme.

Cloning of the dihydroxyacid dehydratase-encoding gene ( ILV3) from Saccharomyces cerevisiae

The biosynthesis of branched-chain amino acids (aa) involves three shared pathways through which pyruvate or α-ketobutyrate are converted into α-keto acids, precursors of valine, leucine or isoleucine. In eukaryotes, few of these common enzymes have been purified to homogeneity, and the whole complement of biosynthetic genes has not been cloned from a single species. In yeasts, most of these genes ( ILV genes) have been cloned and sequenced, with the exception of that coding for dihydroxyacid dehydratase (DAD, EC 4.2.1.9), the third enzyme in the common pathways. We have isolated Saccharomyces cerevisiae genomic sequences by hybridization to an oligodeoxyribonucleotide (oligo) probe designed from a highly conserved domain among bacterial DAD-encoding genes. The cloned sequences have been located to S. cerevisiae chromosome X, mapped within 0.4 centiMorgans (cM) of the ilv3 locus, and found to complement the ilv3 mutations of various yeast strains. Nucleotide (nt) and aa sequence analyses of the longest open reading frame (ORF) located within the cloned sequences identified them as the ILV3 gene, which codes for the yeast DAD. With our cloning of ILV3, yeast becomes the only eukaryotic system from which all ILV genes have been cloned, thus allowing direct molecular analyses of their regulation.

The inactivation of Fe-S cluster containing hydro-lyases by superoxide.

We report in this paper that highly purified Escherichia coli dihydroxy-acid dehydratase, fumarase A, fumarase B, and mammalian aconitase are inactivated by O2- with second order rate constants in the range of 10(6) to 10(7) M-1 s-1. Each of these enzymes belongs to the hydro-lyase class and contains catalytically active [4Fe-4S] clusters. Simultaneous with inactivation by O2- is the release of iron from their clusters. Our working hypothesis is O2- inactivates these enzymes by oxidizing their clusters to an unstable oxidation state, and cluster degradation follows. Consistent with this hypothesis is our observation that spinach dihydroxy-acid dehydratase, a member of the hydro-lyase class that has a catalytically active [2Fe-2S] cluster, is not inactivated and does not lose iron in the presence of O2-. Porcine fumarase, a member of the hydro-lyase class that does not contain an Fe-S cluster, is also not inactivated by O2-. We also report the rate constants for the inactivation of E. coli dihydroxy-acid dehydratase, fumarase A, fumarase B, and mammalian aconitase by O2 are close to 2 x 10(2) M-1 s-1, and the rate constants for the inactivation of E. coli dihydroxy-acid dehydratase and mammalian aconitase by H2O2 are about 10(3) M-1 s-1. E. coli dihydroxy-acid dehydratase has been reported previously to be inactivated in vivo when cells are grown in hyperbaric O2, presumably due to the increased O2- generated under these conditions. We report here that E. coli fumarase A, fumarase B, and aconitase are also inactivated in vivo by hyperbaric O2. Thermodynamic parameters for the oxidation of the cluster of aconitase by O2- and O2 are calculated.

The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase

Dihydroxy-acid dehydratase has been purified from Escherichia coli and characterized as a homodimer with a subunit molecular weight of 66,000. The combination of UV visible absorption, EPR, magnetic circular dichroism, and resonance Raman spectroscopies indicates that the native enzyme contains a [4Fe-4S]2+,+ cluster, in contrast to spinach dihydroxy-acid dehydratase which contains a [2Fe-2S]2+,+ cluster (Flint, D. H., and Emptage, M. H. (1988) J. Biol. Chem. 263, 3558-3564). In frozen solution, the reduced [4Fe-4S]+ cluster has a S = 3/2 ground state with minor contributions from forms with S = 1/2 and possibly S = 5/2 ground states. Resonance Raman studies of the [4Fe-4S]2+ cluster in E. coli dihydroxy-acid dehydratase indicate non-cysteinyl coordination of a specific iron, which suggests that it is likely to be directly involved in catalysis as is the case with aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Münck, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4674-4678). Dihydroxy-acid dehydratase from E. coli is inactivated by O2 in vitro and in vivo as a result of oxidative degradation of the [4Fe-4S]cluster. Compared to aconitase, the oxidized cluster of E. coli dihydroxy-acid dehydratase appears to be less stable as either a cubic or linear [3Fe-4S] cluster or a [2Fe-2S] cluster. Oxidative degradation appears to lead to a complete breakdown of the Fe-S cluster, and the resulting protein cannot be reactivated with Fe2+ and thiol reducing agents.

Near ultraviolet light inactivation of dihydroxyacid dehydratase in Escherichia coli

The effects of near ultraviolet (NUV) light on a NUV chromophore-containing oxidant-sensitive enzyme, dihydroxyacid dehydratase (DHAD), were measured in seven strains of Escherichia coli. The strains differed in production of the oxidant-defense enzymes, superoxide dismutases (Fe-SOD and Mn-SOD), and catalases HPI and HPII. With the stress of aerobic growth but without NUV exposure, the strains lacking either Fe or Mn SOD or both SODs had 57%, 25%, and 12%, respectively, of the DHAD-specific activity of the parent (K12) strain. Under the same conditions, the catalase strains that were wild type, overproducing, and deficient had comparable DHAD-specific activities. When aerobic cultures were exposed for 30 min to NUV with a fluence of 216 J/m 2/s at 310–400 nm, the percentage decreases in DHAD-specific activities were similar (ranging from 75% to 89%) in strains with none, either, or both SODs missing, and in the catalase-overproducing strain. However, the decreases were only 58% and 52% in the strain with catalase missing and in its parent, respectively. The NUV-induced loss of DHAD enzyme activity was not accompanied by any detectable loss of the DHAD protein as measured by polyclonal antibody to DHAD.

Escherichia coli fumarase A catalyzes the isomerization of enol and keto oxalacetic acid.

Fumarase A, a product of the fumA gene of Escherichia coli, has been found to catalyze the isomerization of enol to keto oxalacetic acid (OAA) in addition to catalyzing the fumarase reaction. The kcat/Km for the isomerization is almost identical to that for the fumarase reaction. The isomerization reaction apparently takes place at the same active site as the fumarase reaction since both reactions show a similar sensitivity to inactivation by O2, both reactions are strongly inhibited by 2-hydroxy-3-nitropropionate, and the isomerization reaction is inhibited by fumarate and malate. The isomerization requires the presence of a [4Fe-4S] or [3Fe-4S] cluster, perhaps for structural rather than catalytic reasons. Hydration of enol OAA to the gem diol has been ruled out as a possible mechanism of isomerization on the basis of the preservation of the oxygen on carbon 2 and the position of protonation on carbon 3. The isomerization is not stereospecific in the position of protonation at carbon 3 but appears to be stereoselective, with protonation preferentially occurring in the 3-pro-S position. Porcine fumarase, isopropyl malate isomerase, and dihydroxyacid dehydratase do not catalyze this isomerization. Fumarase A and aconitase, two enzymes with 4Fe-4S clusters that bind a linear 4-carbon dicarboxylic acid moiety in the trans conformation during their normal hydro-lyase reaction, do catalyze this isomerization.

Characterization of enzymes of the branched-chain amino acid biosynthetic pathway in Methanococcus spp

Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae contain similar levels of four enzymes of branched-chain amino acid biosynthesis: acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B. Following growth at low partial pressures of H2-CO2, the levels of these enzymes in extracts of M. voltae are reduced three- to fivefold, which suggests that their synthesis is regulated. The enzymes from M. aeolicus were found to be similar to the eubacterial and eucaryotic enzymes with respect to molecular weights, pH optima, kinetic properties, and sensitivities to O2. The acetohydroxy acid isomeroreductase has a specific requirement for Mg2+, and other divalent cations were inhibitory. It was stimulated threefold by K+ and NH4+ ions and was able to utilize NADH as well as NADPH. The partially purified enzyme was not sensitive to O2. The dihydroxy acid dehydratase is extremely sensitive to O2, and it has a half-life under 5% O2 of 6 min at 25 degrees C. Divalent cations were required for activity, and Mg2+, Mn2+, Ni2+, Co2+, and Fe2+ were nearly equally effective. In conclusion, the archaebacterial enzymes are functionally homologous to the eubacterial and eucaryotic enzymes, which implies that this pathway is very ancient.

Isolation and characterization of ilvA, ilvBN, and ilvD mutants of Caulobacter crescentus

Caulobacter crescentus strains requiring isoleucine and valine (ilv) for growth were shown by transduction and pulsed-field gel electrophoresis to contain mutations at one of two unlinked loci, ilvB and ilvD. Other C. crescentus strains containing mutations at a third locus, ilvA, required either isoleucine or methionine for growth. Biochemical assays for threonine deaminase, acetohydroxyacid synthase, and dihydroxyacid dehydratase demonstrated that the ilvA locus encodes threonine deaminase, the ilvB locus encodes acetohydroxyacid synthase, and the ilvD locus encodes dihydroxyacid dehydratase. C. crescentus strains resistant to the herbicide sulfometuron methyl, which is known to inhibit the action of certain acetohydroxyacid synthases in a variety of bacteria and plants, were shown to contain mutations at the ilvB locus, further suggesting that an acetohydroxyacid synthase gene resides at this locus. Two recombinant plasmids isolated in our laboratory, pPLG389 and pJCT200, were capable of complementing strains containing the ilvB and ilvD mutations, respectively. The DNA in these plasmids hybridized to the corresponding genes of Escherichia coli and Serratia marcescens, confirming the presence of ilvB-like and ilvD-like DNA sequences at the ilvB and ilvD loci, respectively. However, no hybridization was observed between any of the other enteric ilv genes and C. crescentus DNA. These results suggest that C. crescentus contains an isoleucine-valine biosynthetic pathway which is similar to the corresponding pathway in enteric bacteria but that only the ilvB and ilvD genes contain sequences which are highly conserved at the DNA level.

Acetolactate Synthase and Ketol-Acid Reductoisomerase: Targets for Herbicides Obtained by Screening and de novo Design

Several major classes of herbicides, discovered by conventional screening techniques, have been found to inhibit the first common enzyme of branched-chain amino acid biosynthesis, acetolactate synthase, as their mode of action. These herbicides seem to bind to an evolutionary vestige of a quinone-binding site, extraneous to the active site, that is present due to the evolutionary history of this enzyme. Besides their herbicidal effect on sensitive plants, these compounds can effect stasis in the growth of bacteria and yeast. Recently is has been reported that an experimental herbicide from Hoechst. Hoe 704. that was discovered by conventional screening techniques, inhibits the second common enzyme of branched-chain amino acid bio- synthesis [Schultz etal., FEBS Lett. 238, 375-378 (1988)]. We have also recently designed novel reaction-intermediate analogs (e.g. N-isopropyl oxalylhydroxamate) that arc exceptionally potent (Ki = 22 pM: half-time for release approximately six days) and selective inhibitors of the second common enzyme, ketol-acid reductoisomerase. Both of these selective inhibitors of the second common enzyme will kill sensitive plants, but will only inhibit the growth (without killing) of bacteria. The effects in bacteria parallel those obtained by mutations in the relevant genes, where loss of either the first or second common enzyme in the pathway gives an organ- ism that is auxotrophic for branched-chain amino acids, but does not result in a conditionally lethal phenotype. Higher plant mutants have only been obtained to date that arc deficient in functional leucine-specific gene products (as yet uncharacterized), threonine deaminase (isoleucine specific), and dihydroxyacid dehydratase (common). The phenotypes of these mutants. at least at the level of cell culture, are similar to those of their bacterial counterparts, in that auxotrophy, but not conditional lethality, is obtained. These results highlight the potential non-equality of the enzymes of branched-chain amino acid biosynthesis as targets in herbicide design.

Dihydroxyacid dehydratase from E. coli contains a superoxide sensitive [4Fe4S] cluster

Dihydroxyacid dehydratase from E. coli contains a superoxide sensitive [4Fe4S] cluster