Acetohydroxyacid Synthases Research Articles

An easy and sensitive assay for acetohydroxyacid synthases based on the simultaneous detection of substrates and products in a single step.

Acetohydroxyacid synthase (AHAS, EC 2.2.1.6) catalyzes the first step in the synthesis of the branched-chain amino acids valine, leucine, and isoleucine, pathways being present in plants and microorganisms, but not in animals. Thus, AHAS is an important target for numerous herbicides and, more recently, for the development of antimicrobial agents. The need to develop new and optimized herbicides and pharmaceuticals requires a detailed understanding of the biochemistry of AHAS. AHAS transfers an activated two-carbon moiety derived from pyruvate to either pyruvate or 2-oxobutyrate as acceptor substrates, forming 2-acetolactate or 2-acetohydroxy-2-butyrate, respectively. Various methods have been described in the literature to biochemically characterize AHAS with respect to substrate preferences, substrate specificity, or kinetic parameters. However, the simultaneous detection and quantification of substrates and unstable products of the AHAS-catalyzed reaction have always been a challenge. Using AHAS isoform II from Escherichia coli, we have developed a sensitive assay for AHAS-catalyzed reactions that uses derivatization with ethyl chloroformate to stabilize and volatilize all reactants in the aqueous solution and detect them by gas chromatography coupled to flame ionization detection or mass spectrometry. This assay allows us to characterize the product formation in reactions in single and dual substrate reactions and the substrate specificity of AHAS, and to reinterpret previous biochemical observations. This assay is not limited to the AHAS-catalyzed reactions, but should be applicable to studies of many metabolic pathways.

Creation and identification of peanut germplasm tolerant to triazolopyrimidine herbicides

<p id="C3">At present, the germplasm resources of herbicide-tolerant peanut (<italic>Arachis hypogaea</italic>) are scarce, which restrict the diversification of peanut-based cropping system. To create peanut germplasm with tolerance to different herbicides, a mutant population consisting of more than 55,000 peanut lines were generated by ethylmethanesulfonate mutagenesis. We screened this population with a variety of different herbicides to obtain multiple mutants with tolerance to different herbicides. One of these lines displayed strong tolerance to florasulam and flumetsulam in the experiments with foliar herbicide spraying under field conditions as well as in various laboratory evaluation for the herbicide tolerance, while the herbicide-tolerant trait did not have adverse effects on peanut yield and quality. To determine whether this trait was associated with a target-site-based resistance to the herbicides, we compared gene sequences and relative expression levels of these two acetohydroxyacid synthases (AHASs) as the herbicide target enzymes between the mutant and wild type. Molecular cloning and sequence analysis revealed that peanut chromosome A10 and B10 each contained a gene, named as <italic>AhAHAS1a </italic>and <italic>AhAHAS1b</italic>, which were highly similar to <italic>Arabidopsis AHAS</italic>. Peanut chromosome A08 and B08 also each carried an <italic>AHAS</italic> gene, named as <italic>AhAHAS2a </italic>and <italic>AhAHAS2b</italic>, respectively. However, compared with the wild type, the genes in the mutants had no nucleotide substitutions that could alter the amino acid sequences. Furthermore, it was evident that there was no significant difference in the relative expression levels of <italic>AhAHAS</italic> genes between the mutant and wild type. In summary, these results indicate that the herbicide tolerance of the mutant might be caused by non-target-site-based resistance mechanism.

Structures of fungal and plant acetohydroxyacid synthases.

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, is a flavin adenine dinucleotide-, thiamine diphosphate- and magnesium-dependent enzyme that catalyses the first step in the biosynthesis of branched-chain amino acids1. It is the target for more than 50 commercial herbicides2. AHAS requires both catalytic and regulatory subunits for maximal activity and functionality. Here we describe structures of the hexadecameric AHAS complexes of Saccharomyces cerevisiae and dodecameric AHAS complexes of Arabidopsis thaliana. We found that the regulatory subunits of these AHAS complexes form a core to which the catalytic subunit dimers are attached, adopting the shape of a Maltese cross. The structures show how the catalytic and regulatory subunits communicate with each other to provide a pathway for activation and for feedback inhibition by branched-chain amino acids. We also show that the AHAS complex of Mycobacterium tuberculosis adopts a similar structure, thus demonstrating that the overall AHAS architecture is conserved across kingdoms.

Interactions between the ACT Domains and Catalytic Subunits of Acetohydroxyacid Synthases (AHASs) from Different Species.

Acetohydroxyacid synthase (AHAS), which catalyzes the first step in the biosynthesis of branched-chain amino acids, is a target of several types of potent herbicides and antimicrobials. AHAS contains the catalytic subunit (CS) and the regulatory subunit (RS). The AHAS RS is usually composed of ACT domains and C-terminal domains. Herein, it is reported that the ACT domain of AHAS RS from different species could efficiently activate its respective CS. Moreover, the universal cross-activation between the CSs and the ACT domains of RSs across species has been discovered. Based on these biochemical and structural analyses, a molecular basis for the universal ACT-triggered CS activation is proposed, which would help to design broad-spectrum herbicides by targeting the interaction interface between CS and ACT from different species.

Mechanisms of bacterial acetohydroxyacid synthase (AHAS) and specific inhibitors of Mycobacterium tuberculosis AHAS as potential drug candidates against tuberculosis.

On account of the ever increasing resistance of M.tuberculosis strains to orthodox therapy regimens, the task of combating tuberculosis becomes even more challenging. Therefore, there arises a need to isolate new drug targets and subsequently design specific inhibitors for the same. In bacteria, algae, plants and fungi, the synthesis of Branched Chain Amino Acids (BCAAs) is catalyzed by Acetohydroxyacid Synthases (AHAS) group of enzymes. Bacterial AHAS (EC 2.2.1.6) catalyzes the biosynthesis of isoleucine, leucine and valine by utilizing cofactors like Thiamin Diphosphate (ThDP), Flavin Adenine Dinucleotide (FAD) and a divalent metal cation (Usually Mg(2+)). The anabolic form of the enzyme which is presently under discussion consists of two subunits out of which one is catalytic while the other is regulatory in nature. The product of this enzyme catalyzed reaction is either 2-acetolactate or 2-aceto-2-hydroxybutyrate obtained from self-condensation of pyruvate or condensation of puruvate and 2-ketobutyrate, respectively. These are further converted to the BCAAs by a series of other enzymes. The step catalyzed by AHAS is the first in the entire cascade and hence can be selectively targeted for the inhibition of this pathway. M.tuberculosis AHAS, which is encoded by the ilvB and ilvN operons is structurally related to E.coli AHAS and has a similar function. Therefore, specific drugs belonging to the classes of sulfonylureas, imidazolinones and benzoyl esters can be used as inhibitors of M.tuberculosis AHAS which would consequently deplete the BCAA supply to the bacteria. Thus, efficient bacteriostasis can be achieved.

The Minimum Activation Peptide from ilvH Can Activate the Catalytic Subunit of AHAS from Different Species

Acetohydroxyacid synthases (AHASs), which catalyze the first step in the biosynthesis of branched-chain amino acids, are composed of a catalytic subunit (CSU) and a regulatory subunit (RSU). The CSU harbors the catalytic site, and the RSU is responsible for the activation and feedback regulation of the CSU. Previous results from Chipman and co-workers and our lab have shown that heterologous activation can be achieved among isozymes of Escherichia coli AHAS. It would be interesting to find the minimum peptide of ilvH (the RSU of E. coli AHAS III) that could activate other E. coli CSUs, or even those of ## species. In this paper, C-terminal, N-terminal, and C- and N-terminal truncation mutants of ilvH were constructed. The minimum peptide to activate ilvI (the CSU of E. coli AHAS III) was found to be ΔN 14-ΔC 89. Moreover, this peptide could not only activate its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana plumbaginifolia. However, this peptide totally lost its ability for feedback regulation by valine, thus suggesting different elements for enzymatic activation and feedback regulation. Additionally, the apparent dissociation constant (Kd ) of ΔN 14-ΔC 89 when binding CSUs of different species was found to be 9.3-66.5 μM by using microscale thermophoresis. The ability of this peptide to activate different CSUs does not correlate well with its binding ability (Kd ) to these CSUs, thus implying that key interactions by specific residues is more important than binding ability in promoting enzymatic reactions. The high sequence similarity of the peptide ΔN 14-ΔC 89 to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs.

Biomacromolecular 3D‐QSAR to Decipher Molecular Herbicide Resistance in Acetohydroxyacid Synthases

Biomacromolecular 3D‐QSAR to Decipher Molecular Herbicide Resistance in Acetohydroxyacid Synthases

A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

BackgroundStandard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented for several protein families, including lactamases and antibodies, whereas a numbering scheme for the structural family of thiamine-diphosphate (ThDP) -dependent decarboxylases, a large subfamily of the class of ThDP-dependent enzymes encompassing pyruvate-, benzoylformate-, 2-oxo acid-, indolpyruvate- and phenylpyruvate decarboxylases, benzaldehyde lyase, acetohydroxyacid synthases and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD) is still missing.Despite a high structural similarity between the members of the ThDP-dependent decarboxylases, their sequences are diverse and make a pairwise sequence comparison of protein family members difficult.ResultsWe developed and validated a standard numbering scheme for the family of ThDP-dependent decarboxylases. A profile hidden Markov model (HMM) was created using a set of representative sequences from the family of ThDP-dependent decarboxylases. The pyruvate decarboxylase from S. cerevisiae (PDB: 2VK8) was chosen as a reference because it is a well characterized enzyme. The crystal structure with the PDB identifier 2VK8 encompasses the structure of the ScPDC mutant E477Q, the cofactors ThDP and Mg2+ as well as the substrate analogue (2S)-2-hydroxypropanoic acid. The absolute numbering of this reference sequence was transferred to all members of the ThDP-dependent decarboxylase protein family. Subsequently, the numbering scheme was integrated into the already established Thiamine-diphosphate dependent Enzyme Engineering Database (TEED) and was used to systematically analyze functionally and structurally relevant positions in the superfamily of ThDP-dependent decarboxylases.ConclusionsThe numbering scheme serves as a tool for the reliable sequence alignment of ThDP-dependent decarboxylases and the unambiguous identification and communication of corresponding positions. Thus, it is the basis for the systematic and automated analysis of sequence-encoded properties such as structural and functional relevance of amino acid positions, because the analysis of conserved positions, the identification of correlated mutations and the determination of subfamily specific amino acid distributions depend on reliable multisequence alignments and the unambiguous identification of the alignment columns. The method is reliable and robust and can easily be adapted to further protein families.

Interactions between Large and Small Subunits of Different Acetohydroxyacid Synthase Isozymes of Escherichia coli

The large, catalytic subunits (LSUs; ilvB, ilvG and ilvI, respectively) of enterobacterial acetohydroxyacid synthases isozymes (AHAS I, II and III) have molecular weights approximately 60 kDa and are paralogous with a family of other thiamin diphosphate dependent enzymes. The small, regulatory subunits (SSUs) of AHAS I and AHAS III (ilvN and ilvH) are required for valine inhibition, but ilvN and ilvH can only confer valine sensitivity on their own LSUs. AHAS II is valine resistant. The LSUs have only approximately 15, <<1 and approximately 3%, respectively, of the activity of their respective holoenzymes, but the holoenzymes can be reconstituted with complete recovery of activity. We have examined the activation of each of the LSUs by SSUs from different isozymes and ask to what extent such activation is specific; that is, is effective nonspecific interaction possible between LSUs and SSUs of different isozymes? To our surprise, the AHAS II SSU ilvM is able to activate the LSUs of all three of the isozymes, and the truncated AHAS III SSUs ilvH-Delta80, ilvH-Delta86 and ilvH-Delta89 are able to activate the LSUs of both AHAS I and AHAS III. However, none of the heterologously activated enzymes have any feedback sensitivity. Our results imply the existence of a common region in all three LSUs to which regulatory subunits may bind, as well as a similarity between the surfaces of ilvM and the other SSUs. This surface must be included within the N-terminal betaalphabetabetaalphabeta-domain of the SSUs, probably on the helical face of this domain. We suggest hypotheses for the mechanism of valine inhibition, and reject one involving induced dissociation of subunits.

Origin of the specificities of acetohydroxyacid synthases and glyoxylate carboligase

Acetohydroxyacid synthases (AHAS) and glyoxylate carboligase (GCL) catalyze decarboxylation of 2-ketoacids and condensation of the resulting hydroxyalkylThDP anions/enamines with a second ketoacid to form 2-acyl-2-hydroxyacids. AHASs prefer pyruvate by >10-fold over any other ketoacid as first substrate. Steric hindrance seems to be the major determinant of this specificity; Escherichia coli AHAS isozyme II mutant Val375Ala allows 2-ketobutyrate (C 2H 5COCO 2 −) to be a good first substrate and the mutant enzyme can thus synthesize 2-propio-2-hydroxybutyrate. An Ile residue in the equivalent position in GCL (Ile393) may play the analogous role in restricting GCL to glyoxylate (HCOCO 2 −) as first substrate. The specificity of AHAS for 2-ketoacids as acceptor substrates is due to an arginine residue which probably interacts with the carboxylate of the second substrate ( e.g., Arg276 in AHASII). Mutants altered at this arginine can utilize aromatic aldehydes as second substrate and form chiral arylacyl carbinols, of interest as precursors for pharmaceutical syntheses. Analysis of AHAS II supports a mechanism in which carboligation occurs after rate-determining formation of hydroxyethylThDP. NMR measurements of the distribution of ThDP-bound intermediates showed that a faster rate constant for product release when the alkyl group derived from the acceptor substrate is ethyl compared to methyl plays a major role in product specificity. The crucial role of a Trp residue (Trp 464 in AHASII) in determining specificity may be due to control of a conformational change involved in product release rather than to affinity for 2-ketobutyrate. It is significant that in AHAS I, without the required Trp and with a low specificity for 2-ketobutyrate as acceptor substrate, the product release step is rapidly reversible.

Deregulation of acetohydroxy-acid synthase: Loss of allosteric inhibition conferred by mutations in the catalytic subunit

Acetohydroxy-acid synthases (AHAS) of two mutant strains Streptomyces cinnamonensis ACB-NLR-2 and BVR-18 were chosen for this study for their apparent activation by valine, which regularly acts as an allosteric inhibitor. Sequencing the ilvB genes coding for the AHAS catalytic subunit revealed two distant changes in the mutants, DeltaQ217 and E139A, respectively. Homology modeling was used to propose the structural changes caused by those mutations. In the mutant strain ACB-NLR-2 (resistant to 2-amino-3-chlorobutyrate and norleucine), deletion of Q217 affected a helix in ss-domain, distant from the active center. As no mutation was found in the regulatory subunit of this strain, DeltaQ217 in IlvB was supposed to be responsible for the observed valine activation, probably via changed properties on the proposed regulatory-catalytic subunit interface. In mutant strain BVR-18 (resistant to 2-oxobutyrate), substitution E139A occurred in a conservative loop near the active center. In vitro AHAS activity assay with the enzyme reconstituted from the wild-type regulatory and BVR-18 catalytic subunits proved that the substitution in the catalytic subunit led to the apparent activation of AHAS by valine. We suggest that the conservative loop participated in a conformational change transfer to the active center during the allosteric regulation.

Overproduction of noncanonical amino acids by Escherichia coli cells

Overproduction of noncanonical amino acids norvaline and norleucine by Escherichia coli with inactivated acetohydroxy acid synthases was demonstrated. The cultivation conditions for the overproduction of noncanonical amino acids were studied. The effect of the restoration of acetohydroxy acid synthase activity, increased expression of the leuABCD operon, and inactivation of the biosynthetic threonine deaminase on norvaline and norleucine synthesis was studied. When grown under valine limitation, E. coli cells with inactivated acetohydroxy acid synthases and an elevated level of expression of the valine operon were shown to accumulate norvaline and norleucine (up to 0.8 and 4 g/l, respectively). These results confirm the existing hypothesis of norvaline and norleucine formation from 2-ketobutyrate by leucine biosynthesis enzymes.

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

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

Binding and activation of thiamin diphosphate in acetohydroxyacid synthase.

Acetohydroxyacid synthases (AHASs) are biosynthetic thiamin diphosphate- (ThDP) and FAD-dependent enzymes. They are homologous to pyruvate oxidase and other members of a family of ThDP-dependent enzymes which catalyze reactions in which the first step is decarboxylation of a 2-ketoacid. AHAS catalyzes the condensation of the 2-carbon moiety, derived from the decarboxylation of pyruvate, with a second 2-ketoacid, to form acetolactate or acetohydroxybutyrate. A structural model for AHAS isozyme II (AHAS II) from Escherichia coli has been constructed on the basis of its homology with pyruvate oxidase from Lactobacillus plantarum (LpPOX). We describe here experiments which further test the model, and test whether the binding and activation of ThDP in AHAS involve the same structural elements and mechanism identified for homologous enzymes. Interaction of a conserved glutamate with the N1' of the ThDP aminopyrimidine moiety is involved in activation of the cofactor for proton exchange in several ThDP-dependent enzymes. In accord with this, the analogue N3'-pyridyl thiamin diphosphate does not support AHAS activity. Mutagenesis of Glu47, the putative conserved glutamate, decreases the rate of proton exchange at C-2 of bound ThDP by nearly 2 orders of magnitude and decreases the turnover rate for the mutants by about 10-fold. Mutant E47A also has altered substrate specificity, pH dependence, and other changes in properties. Mutagenesis of Asp428, presumed on the basis of the model to be the crucial carboxylate ligand to Mg(2+) in the "ThDP motif", leads to a decrease in the affinity of AHAS II for Mg(2+). While mutant D428N shows ThDP affinity close to that of the wild-type on saturation with Mg(2+), D428E has a decreased affinity for ThDP. These mutations also lead to dependence of the enzyme on K(+). These experiments demonstrate that AHAS binds and activates ThDP in the same way as do pyruvate decarboxylase, transketolase, and other ThDP-dependent enzymes. The biosynthetic activity of AHAS also involves many other factors beyond the binding and deprotonation of ThDP; changes in the ligands to ThDP can have interesting and unexpected effects on the reaction.

Regulation by external pH and stationary growth phase of the acetolactate synthase from Synechocystis PCC6803.

Several characteristics identify the protein encoded by the alsS gene [sll1981 in Cyanobase (http://www.kazusa.or.jp/cyano/cyano. html)] of Synechocystis PCC6803 as an acetolactate synthase. The AlsS protein is about 60% homologous to the AlsS from Bacillus subtilis or other bacteria. These enzymes condense two pyruvates to form acetolactate, implicated in pH homeostasis via the acetoin-2, 3-butanediol pathway or in valine biosynthesis. Transcriptional fusions revealed that alsS was induced at the onset of stationary phase, as in B. subtilis, a situation leading to an increase in the pHout to above 11 in Synechocystis. This is the first cyanobacterial gene showing a dependence on pH for its expression. Induction was also obtained by the presence of > 100 mM Na+, the effect being prevented by amiloride, in agreement with Na+/H+ exchange in the pH homeostasis process. Homology of the Synechocystis AlsS protein to the close family of acetohydroxy acid synthases (including one in Synechocystis) is around 30%. These enzymes are involved in the parallel routes for valine/leucine and isoleucine biosynthesis. No phenotype of auxotrophy for any of these amino acids was associated with a null mutation in the Synechocystis alsS gene. The AlsS enzyme did not complement the isoleucine deficiency of an acetohydroxy acid synthase-deficient Escherichia coli mutant.

Deletion of the pyc gene blocks clavulanic acid biosynthesis except in glycerol-containing medium: evidence for two different genes in formation of the C3 unit.

The beta-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster of Streptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of the pyc gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the pyc gene with the aph gene did not cause isoleucine-valine auxotrophy in the mutant. The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the beta-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing D-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. The pyc replacement mutant overproduces cephamycin.

Channeling Behavior and Activity Models for Escherichia coli K-12 Acetohydroxy Acid Synthases at Physiological Substrate Levels

The channeling behavior of acetohydroxy acid synthases I and III (EC 4.1.3.18; AHAS) was studied by computer simulation of activities over a wide range of concentrations for the substrates pyruvate and 2-ketobutyrate. The ratios of reaction rates for both channels and three-dimensional plots of single-channel reaction rates versus substrate concentrations were introduced to compare the substrate channeling properties of the isozymes. Substrate ranges were identified in which AHAS I and III operated both channels, and in which they used only one. Kinetic constants were varied to simulate whether and how AHAS might be made channel-specific. Our study suggests that AHAS might be made channel-specific for acetolactate but not for acetohydroxybutyrate. We postulate specific physiological roles for AHAS I and III to support cell growth under conditions that vary the levels and balance of substrates.

Molecular cloning, DNA sequencing, and biochemical analyses of Escherichia coli glyoxylate carboligase. An enzyme of the acetohydroxy acid synthase-pyruvate oxidase family.

Glyoxylate carboligase (Gcl) (EC 4.1.1.47) of Escherichia coli catalyzes the condensation of two molecules of glyoxylate to give tartronic semialdehyde, a key intermediate in glyoxylate catabolism. We report the cloning, genomic location, and DNA sequence of the gene (called gcl) encoding E. coli Gcl and isolation of mutants lacking the enzyme. Gcl is a protein of 593 amino acid residues (64,738 Da) that has a high level (30%) of sequence similarity to the acetohydroxy acid synthases (AHAS) of branched chain amino acid synthetic pathway. Significant sequence identity (26%) was also observed with E. coli pyruvate oxidase, a redox flavoprotein, previously shown to be related to the AHAS enzymes (Chang, Y.-Y., and Cronan, J. E., Jr. (1988) J. Bacteriol. 170, 3937-3945). Consistent with a grouping of Gcl with the AHAS and pyruvate oxidase enzymes. Gcl contains a quinone binding site as well as binding site for thiamine pyrophosphate and FAD. We also found that a gene (orf258) immediately downstream of the gcl gene encoded a protein (Orf258) of 258 residues. Although the gene organization of gcl and orf258 is analogous to that of the ilv gene operons which encode the E. coli AHAS isozyme large and small subunits, Orf258 does not function as a Gcl subunit. Moreover, disruption of the chromosomal copy of orf258 did not affect growth on glyoxylate or glycolate.

Properties of subcloned subunits of bacterial acetohydroxy acid synthases.

The acetohydroxy acid synthase (AHAS) isozymes from enterobacteria are each composed of a large and small subunit in an alpha 2 beta 2 structure. It has been generally accepted that the large (ca. 60-kDa) subunits are catalytic, while the small ones are regulatory. In order to further characterize the roles of the subunits as well as the nature and the specificities of their interactions, we have constructed plasmids encoding the large or small subunits of isozymes AHAS I and AHAS III, each with limited remnants of the other peptide. The catalytic properties of the large subunits have been characterized and compared with those of extracts containing the intact enzyme or of purified enzymes. Antisera to the isolated subunits have been used in Western blot (immunoblot) analyses for qualitative and semiquantitative determinations of the presence of the polypeptides in extracts. The large subunits of AHAS isozymes I and III have lower activities than the intact enzymes: Vmax/Km is 20 to 50 times lower in both cases. However, for AHAS I, most of this difference is due to the raised Km of the large subunit alone, while for AHAS III, it is due to a lowered Vmax. The substrate specificities, R, of large subunits are close to those of the intact enzymes. The catalytic activity of the large subunits of AHAS I is dependent on flavin adenine dinucleotide (FAD), as is that of the intact enzyme, although the apparent affinities of the large subunits alone for FAD are 10-fold lower. Isolated subunits are insensitive to valine inhibition. Nearly all of the properties of the intact AHAS isozyme I or III can be reconstituted by mixing extracts containing the respective large and small subunits. The mixing of subunits from different enzymes does not lead to activation of the large subunits. It is concluded that the catalytic machinery of these AHAS isozymes is entirely contained within the large subunits. The small subunits are required, however, for specific stabilization of an active conformation of the large subunits as well as for value sensitivity.

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.