A Novel Antibiotic Mechanism of l-Cyclopropylalanine Blocking the Biosynthetic Pathway of Essential Amino Acid l-Leucine.

The unusual amino acid l-cyclopropylalanine was isolated from the mushroom Amanita virgineoides after detection in an anti-fungal screening test. l-Cyclopropylalanine was found to exhibit broad-spectrum inhibition against fungi and bacteria. The anti-fungal activity was found to be abolished in the presence of the amino acid l-leucine, but not any other amino acids, indicating that l-cyclopropylalanine may block the biosynthesis of the essential amino acid l-leucine, thereby inhibiting fungal and bacteria growth. Further biochemical studies found l-cyclopropylalanine indeed inhibits α-isopropylmalate synthase (α-IMPS), the enzyme that catalyzes the rate-limiting step in the biosynthetic pathway of l-leucine. Inhibition of essential l-leucine synthesis in fungal and bacteria organisms, a pathway absent in host organisms such as humans, may represent a novel antibiotic mechanism to counter the ever-increasing problem of drug resistance to existing antibiotics.

Evolutionarily distinct versions of the multidomain enzyme α-isopropylmalate synthase share discrete mechanisms of V-type allosteric regulation.

Understanding the evolution of allostery in multidomain enzymes remains an important step in improving our ability to identify and exploit structure-function relationships in allosteric mechanisms. A recent protein similarity network for the DRE-TIM metallolyase superfamily indicated there are two evolutionarily distinct forms of the enzyme α-isopropylmalate synthase (IPMS) sharing approximately 20% sequence identity. IPMS from Mycobacterium tuberculosis has been extensively characterized with respect to catalysis and the mechanism of feedback regulation by l-leucine. Here, IPMS from Methanococcus jannaschii (MjIPMS) is used as a representative of the second form of the enzyme, and its catalytic and regulatory mechanism is compared with that of MtIPMS to identify any functional differences between the two forms. MjIPMS exhibits kinetic parameters similar to those of other reported IPMS enzymes and is partially inhibited by l-leucine in a V-type manner. Identical values of (D2O)kcat (3.1) were determined in the presence and absence of l-leucine, indicating the hydrolytic step is rate-determining in the absence of l-leucine and remains so in the inhibited form of the enzyme. This mechanism is identical to the mechanism identified for MtIPMS ((D2O)kcat = 3.3 ± 0.3 in the presence of l-leucine) despite product release being rate-determining in the uninhibited MtIPMS enzyme. The identification of identical regulatory mechanisms in enzymes with low sequence identity raises important evolutionary questions concerning the acquisition and divergence of multidomain allosteric enzymes and highlights the need for caution when comparing regulatory mechanisms for homologous enzymes.

Mechanistic and bioinformatic investigation of a conserved active site helix in α-isopropylmalate synthase from Mycobacterium tuberculosis, a member of the DRE-TIM metallolyase superfamily.

The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily.

V-type allosteric inhibition is described by a shift in the rate-determining step for α-isopropylmalate synthase from Mycobacterium tuberculosis.

The kinetic parameters affected by allosteric mechanisms contain collections of rate constants that vary based on differences in the relative rates of individual steps in the reaction. Thus, it may not be useful to compare enzymes with similar allosteric mechanisms unless the point of regulation has been identified. Rapid reaction kinetics and kinetic isotope effects provide a detailed description of V-type feedback allosteric inhibition in α-isopropylmalate synthase from Mycobacterium tuberculosis, an evolutionarily conserved model allosteric system. Results are consistent with a shift in the rate-determining step from product release to the hydrolytic step in catalysis in the presence of the effector.

Amino-acid substitutions at the domain interface affect substrate and allosteric inhibitor binding in α-isopropylmalate synthase from Mycobacterium tuberculosis.

α-Isopropylmalate synthase (α-IPMS) is a multi-domain protein catalysing the condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate. This reaction is the first committed step in the leucine biosynthetic pathway in bacteria and plants, and α-IPMS is allosterically regulated by this amino acid. Existing crystal structures of α-IPMS from Mycobacterium tuberculosis (MtuIPMS) indicate that this enzyme has a strikingly different domain arrangement in each monomer of the homodimeric protein. This asymmetry results in two distinct interfaces between the N-terminal catalytic domains and the C-terminal regulatory domains in the dimer. In this study, residues Arg97 and Asp444 across one of these unequal domain interfaces were substituted to evaluate the importance of protein asymmetry and salt bridge formation between this pair of residues. Analysis of solution-phase structures of wild-type and variant MtuIPMS indicates that substitutions of these residues have little effect on overall protein conformation, a result also observed for addition of the feedback inhibitor leucine to the wild-type enzyme. All variants had increased catalytic efficiency relative to wild-type MtuIPMS, and those with an Asp444 substitution displayed increased affinity for the substrate AcCoA. All variants also showed reduced sensitivity to leucine and altered biphasic reaction kinetics when compared with those of the wild-type enzyme. It is proposed that substituting residues at the asymmetric domain interface increases flexibility in the protein, particularly affecting the AcCoA binding site and the response to leucine, without penalty on catalysis.

The slow-onset nature of allosteric inhibition in α-isopropylmalate synthase from Mycobacterium tuberculosis is mediated by a flexible loop.

The identification of structure-function relationships in allosteric enzymes is essential to describing a molecular mechanism for allosteric processes. The enzyme α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) is subject to slow-onset, allosteric inhibition by l-leucine. Here we report that alternate amino acids act as rapid equilibrium noncompetitive inhibitors of MtIPMS failing to display biphasic inhibition kinetics. Amino acid substitutions on a flexible loop covering the regulatory binding pocket generate enzyme variants that have significant affinity for l-leucine but lack biphasic inhibition kinetics. Taken together, these results are consistent with the flexible loop mediating the slow-onset step of allosteric inhibition.

The C-terminal regulatory domain is required for catalysis by Neisseria meningitidis alpha-isopropylmalate synthase.

alpha-Isopropylmalate synthase (alpha-IPMS) catalyses the first committed step in leucine biosynthesis in many pathogenic bacteria, including Neisseria meningitidis. This enzyme (NmeIPMS) has been purified, characterised, and compared to alpha-IPMS proteins from other bacteria. NmeIPMS is a homodimer which catalyses the condensation of alpha-ketoisovalerate (alpha-KIV) and acetyl coenzyme A (AcCoA), and is inhibited by leucine. NmeIPMS can use alternate alpha-ketoacids as substrates and, in contrast to alpha-IPMS from other sources, is activated by a range of metal ions including Cd(2+) and Zn(2+) that have previously been reported as inhibitory, since they suppress the dithiodipyridone assay system rather than the enzyme itself. Previous studies indicate that alpha-IPMS is a TIM barrel enzyme with an allosteric leucine-binding domain. To assess the importance of this domain, a truncated form of NmeIPMS was generated and characterised. Loss of the regulatory domain resulted in a loss of the ability to catalyse the aldol reaction, although the enzyme was still able to slowly hydrolyse AcCoA independently of alpha-KIV at a rate similar to that of the WT enzyme. This implies that the regulatory domain is not only required for control of enzymatic activity but may assist in the positioning of key residues in the catalytic TIM barrel. The importance of this domain to catalytic function may offer new strategies for inhibitor design.

Mapping of the allosteric network in the regulation of alpha-isopropylmalate synthase from Mycobacterium tuberculosis by the feedback inhibitor L-leucine: solution-phase H/D exchange monitored by FT-ICR mass spectrometry.

As it is becoming accepted that allosteric regulation can occur through a change in local conformational equilibria as opposed to a change in overall static structure, a thorough description of the structural aspects of these types of mechanisms will be essential to understanding this fundamental biological process. Here we report the experimental identification of key regions of conformational perturbation in the allosteric network of a large (144 kDa), multidomain enzyme by use of solution-phase hydrogen/deuterium exchange. Large perturbations in the regulatory domain induced by effector molecule binding are linked to a very specific, targeted perturbation in the active site, some 50 A away. Binding of L-leucine to an enzyme variant (Y410F) that is kinetically insensitive to effector binding was shown to elicit similar changes in the regulatory domain, but perturbs an alternate region of the catalytic domain, consistent with the proposed allosteric mechanism. These results comprise one of the first reports of an experimentally mapped allosteric mechanism in a protein of this size and provide necessary information to be used toward the development of allostery-based drugs or enzymes with engineered regulatory properties.

Kinetic and chemical mechanism of alpha-isopropylmalate synthase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis alpha-isopropylmalate synthase (MtIPMS) catalyzes the condensation of acetyl-coenzyme A (AcCoA) with alpha-ketoisovalerate (alpha-KIV) and the subsequent hydrolysis of alpha-isopropylmalyl-CoA to generate the products CoA and alpha-isopropylmalate (alpha-IPM). This is the first committed step in l-leucine biosynthesis. We have purified recombinant MtIPMS and characterized it using a combination of steady-state kinetics, isotope effects, isotopic labeling, and (1)H-NMR spectroscopy. The alpha-keto acid specificity of the enzyme is narrow, and the acyl-CoA specificity is absolute for AcCoA. In the absence of alpha-KIV, MtIPMS does not enolize the alpha protons of AcCoA but slowly hydrolyzes acyl-CoA analogues. Initial velocity studies, product inhibition, and dead-end inhibition studies indicate that MtIPMS follows a nonrapid equilibrium random bi-bi kinetic mechanism, with a preferred pathway to the ternary complex. MtIPMS requires two catalytic bases for maximal activity (both with pK(a) values of ca. 6.7), and we suggest that one catalyzes deprotonation and enolization of AcCoA and the other activates the water molecule involved in the hydrolysis of alpha-isopropylmalyl-CoA. Primary deuterium and solvent kinetic isotope effects indicate that there is a step after chemistry that is rate-limiting, although, with poor substrates such as pyruvate, hydrolysis becomes partially rate-limiting. Our data is inconsistent with the suggestion that a metal-bound water is involved in hydrolysis. Finally, our results indicate that the hydrolysis of alpha-isopropylmalyl-CoA is direct, without the formation of a cyclic anhydride intermediate. On the basis of these results, a chemical mechanism for the MtIPMS-catalyzed reaction is proposed.

Slow-onset feedback inhibition: inhibition of Mycobacterium tuberculosis alpha-isopropylmalate synthase by L-leucine.

This report describes the first demonstration of slow-onset feedback inhibition of an enzyme that catalyzes the first committed step in a biosynthetic pathway. alpha-Isopropylmalate synthase (IPMS) catalyzes the first committed step of the l-leucine biosynthetic pathway and is feedback-inhibited by l-leucine. Initial velocity experiments on the Mycobacterium tuberculosis IPMS indicate that inhibition by l-leucine is linearly noncompetitive versus alpha-ketoisovalerate. Time-courses displayed a burst of product formation followed by a linear steady-state rate when reactions were initiated by the addition of enzyme. The burst rate showed a hyperbolic dependence on the concentration of l-leucine indicating that inhibition proceeds in two steps, an initial rapid binding step followed by slow isomerization to a more tightly bound complex.

Enhanced formation of isoamyl alcohol in Zygosaccharomyces rouxii due to elimination of feedback inhibition of alpha-isopropylmalate synthase.

Mutants of the 'miso' yeast, Zygosaccharomyces rouxii, that produced a large amount of isoamyl alcohol, an important flavour in miso fermentation, were isolated from 5,5,5-trifluoro-DL-leucine-resistant mutants, an analogue of L-leucine. One of the mutants, M21-10, produced a three-fold higher level of isoamyl alcohol than the wild-type strain MY21 in miso fermentation. The activity of alpha-isopropylamalate synthase, one of the enzymes used for L-leucine synthesis, in the mutant M21-10 was not inhibited by the addition of L-leucine, a feedback inhibitor.

Multiple new genes that determine activity for the first step of leucine biosynthesis in Saccharomyces cerevisiae.

The first step in the biosynthesis of leucine is catalyzed by alpha-isopropylmalate (alpha-IPM) synthase. In the yeast Saccharomyces cerevisiae, LEU4 encodes the isozyme responsible for the majority of alpha-IPM synthase activity. Yeast strains that bear disruption alleles of LEU4, however, are Leu+ and exhibit a level of synthase activity that is 20% of the wild type. To identify the gene or genes that encode this remaining activity, a leu4 disruption strain was mutagenized. The mutations identified define three new complementation groups, designated leu6, leu7 and leu8. Each of these new mutations effect leucine auxotrophy only if a leu4 mutation is present and each results in loss of alpha-IPM synthase activity. Further analysis suggests that LEU7 and LEU8 are candidates for the gene or genes that encode an alpha-IPM synthase activity. The results demonstrate that multiple components determine the residual alpha-IPM synthase activity in leu4 gene disruption strains of S. cerevisiae.

Total deletion of yeast LEU4: further evidence for a second alpha-isopropylmalate synthase and evidence for tight LEU4-MET4 linkage.

Using a combination of restriction endonuclease digestion, nuclease BAL 31 treatment, and standard ligation procedures, a 4.4-kb DNA segment that carried the yeast LEU4 gene [encoding alpha-isopropylmalate synthase (IPMS) I] and adjoining sequences was excised from an appropriate plasmid and replaced with the yeast HIS3 gene. The new plasmid was digested to obtain a linear HIS3-carrying fragment flanked by remnants of the LEU4 region. Integrative transformation of a LEU4fbr LEU5+ his3- strain with this fragment resulted in the deletion of the LEU4 gene from the genome of some recipients, as demonstrated by transformant phenotype, genetic analysis and the absence of RNA capable of hybridizing to a LEU4 probe. The leu4 deletion strains remained Leu+. The extract of one such strain contained about 18% of the IPMS activity of wild-type cells. It is concluded that the residual activity is that of a second IPMS (IPMS II) that depends on an intact LEU5 locus. IPMS II was inhibited by leucine, but its sensitivity was about an order of magnitude lower than that of IPMS I. Deletion of the LEU4 region by the method utilized here resulted in an amino acid auxotrophy that could be satisfied by methionine, homocysteine, or cysteine. Complementation tests and genetic analysis demonstrated that the affected gene was MET4. Linkage to MET4 would place the LEU4 gene on the left arm of chromosome XIV.

Inactivation of yeast alpha-isopropylmalate synthase by CoA. Antagonism between CoA and adenylates and the mechanism of CoA inactivation.

Yeast alpha-isopropylmalate synthase (EC 4.1.3.12) is inactivated by micromolar concentrations of CoA in the presence of Zn2+. We report here that rapid reactivation of inactivated enzyme (full recovery in less than 10 min) occurred in the presence of millimolar concentrations of ATP or ADP, using permeabilized cells. With purified, CoA-inactivated enzyme, ATP had only a weak reactivating effect which increased drastically, however, when a chelator was added at a concentration (0.1 mM) which by itself had little effect. Higher concentrations of chelator (1 mM) caused rapid reactivation even in the absence of ATP. Reactivation was also possible by removing CoA from equilibrium with oxidized glutathione, with acetyl phosphate in the presence of phosphotransacetylase, or by dialysis; however, these processes were very slow. Protection against CoA inactivation of alpha-isopropylmalate synthase was provided by high concentrations of ATP and, to a much lesser extent, ADP, by a high adenylate energy charge, by chelators, and by 3'-dephospho-CoA. Enzyme which had been inactivated with [3H]CoA did not retain any radioactivity (above control) when extracted with phenol. This result, together with other observations, is interpreted to mean that inactivation does not involve covalent modification, but is more likely the result of the formation of an enzyme.CoA.zinc complex held together by noncovalent forces. The physiological significance of the CoA effect is discussed.

alpha-Isopropylmalate synthase from yeast. A zinc metalloenzyme.

Highly purified alpha-isopropylmalate synthase (3-hydroxy-4-methyl-3-carboxyvalerate 2-oxo-3-methylbutyrate-lysase (coA-acetylating), EC 4.1.3.12) from Saccharomyces cerevisiae is inactivated by various chelating agents. Atomic absorption spectrometry indicates that the enzyme contains approx. four gatoms of zinc per dimer of molecular weight of 130 000. Dialysis against ethylenediaminetetraacetic acid at an initial concentration of 0.1 mM reduces the zinc content to about two gatoms of zinc per dimer. While such enzyme remains active, it has altered kinetic properties and is stimulated by Mn2+, in contrast to untreated enzyme. Dialysis against ethylenediaminetetraacetic acid at an initial concentration of 50 mM reduces the zinc content by more than 80% and causes almost complete loss of enzymatic activity. Activity can be restored by the addition of Zn2+, Mn2+, Fe2+, Co2+, or Cd2+.

Alpha-isopropylmalate synthase from Alcaligenes eutrophus H 16. III. Endproduct inhibition and its relief by valine and isoleucine.

The alpha-isopropylmalate synthase (EC 4.1.3.12) from Alcaligenes eutrophus H 16 was inhibited by L-leucine and alpha-ketoisocaproate. The extent of inhibition was influenced by substrate- and inhibitor concentrations as well as by the pH. Intermediary plateaus, which always appeared in the inhibition curves, suggested cooperative effects. The maximal Hill coefficient was found to be two. At low concentrations of leucine the inhibition mechanism was of the competitive type with respect to substrate acetyl coenzyme A and of the noncompetitive type with respect to substrate alpha-ketoisovalerate. The inhibition was specifically relieved by the addition of valine or isoleucine. The anomalous effect of temperature on enzyme activity was diminished by leucine. The Arrhenius energy of the reaction increased from about 11 kcal/mole in the absence of leucine to about 18 kcal/mole in the presence of leucine. The further addition of valine reversed this effect. The physiological relevance of the alpha-ketoisocaproate-mediated inhibition is discussed.

Reversible, coenzyme-A-mediated inactivation of biosynthetic condensing enzymes in yeast: a possible regulatory mechanism.

alpha-Isopropylmalate synthase [3-hydroxy-4-methyl-3-carboxyvalerate 2-oxo-3-methylbutyrate-lyase (CoA-acetylating); EC 4.1.3.12], the enzyme catalyzing the first committed step in leucine biosynthesis, and homocitrate synthase [3-hydroxy-3-carboxyadipate 2-oxoglutarate-lyase (CoA-acetylating); EC 4.1.3.21], the first enzyme in lysine biosynthesis in yeast, are rapidly inactivated in the presence of low concentrations of coenzyme A, a product of both reactions. Closely related compounds like 3-dephospho-coenzyme A or oxidized coenzyme A are almost without effect, as are other sulfhydryl compounds. Citrate (si)-synthase [citrate oxaloacetate-lyase (pro-3S-CH2-COO-minus leads to acetyl-CoA); EC 4.1.3.7] appears to be completely resistant against inactivation by coenzyme A. Inactivated alpha-isopropylmalate and homocitrate synthases can be reactivated by dialysis, but not by adding excess substrate. Protection against coenzyme-A-mediated inactivation is provided by relatively high concentrations of the alpha-ketoacid substrate or the specific end product inhibitor of each of the two enzymes. The coenzyme-A-mediated inactivation of alpha-isopropylmalate synthase has been more closely investigated. It requires the presence of divalent metal ions, with Zn++being most effective. The inactivation does not require molecular oxygen. It occurs in the presence of low concentrations of substrates and is observed in toluene-treated cells. These results, together with evidence that alpha-isopropylmalate synthase and homocitrate synthase are located in the mitochondria, suggest a mechanism by which increasing intra-mitochondrial coenzyme A concentrations might serve as a signal of decreasing acetyl-coenzyme A levels, triggering a temporary inactivation of biosynthetic acetyl-coenzyme A-consuming reactions in order to channel the available acetyl-coenzyme A into the citrate cycle.