Subdomain II of α-Isopropylmalate Synthase Is Essential for Activity: INFERRING A MECHANISM OF FEEDBACK INHIBITION*

The committed step of leucine biosynthesis, converting acetyl-CoA and α-ketoisovalerate into α-isopropylmalate, is catalyzed by α-isopropylmalate synthase (IPMS), an allosteric enzyme subjected to feedback inhibition by the end product l-leucine. We characterized the short form IPMS from Leptospira biflexa (LbIPMS2), which exhibits a catalytic activity comparable with that of the long form IPMS (LbIPMS1) and has a similar N-terminal domain followed by subdomain I and subdomain II but lacks the whole C-terminal regulatory domain. We found that partial deletion of the regulatory domain of LbIPMS1 resulted in a loss of about 50% of the catalytic activity; however, when the regulatory domain was deleted up to Arg-385, producing a protein that is almost equivalent to the intact LbIPMS2, about 90% of the activity was maintained. Moreover, in LbIPMS2 or LbIPMS1, further deletion of several residues from the C terminus of subdomain II significantly impaired or completely abolished the catalytic activity, respectively. These results define a complete and independently functional catalytic module of IPMS consisting of both the N-terminal domain and the two subdomains. Structural comparison of LbIPMS2 and the Mycobacterium tuberculosis IPMS revealed two different conformations of subdomain II that likely represent two substrate-binding states related to cooperative catalysis. The biochemical and structural analyses together with the previously published hydrogen-deuterium exchange data led us to propose a conformation transition mechanism for feedback inhibition mediated by subdomains I and II that might associated with alteration of the binding affinity toward acetyl-CoA.     

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.     

Modifying the determinants of α-ketoacid substrate selectivity in mycobacterium tuberculosis α-isopropylmalate synthase

α-Isopropylmalate synthase (IPMS) catalyses the reaction between α-ketoisovalerate and acetyl coenzyme A (AcCoA) in the first step of leucine biosynthesis. IPMS is closely related to homocitrate synthase, which catalyses the reaction between AcCoA and the unbranched α-ketoacid α-ketoglutarate. Analysis of these enzymes suggests that several differently conserved key residues are responsible for the different substrate selectivity. These residues were systematically substituted in the Mycobacterium tuberculosis IPMS, resulting in changes in substrate specificity. A variant of IPMS was constructed with a preference for the unbranched α-ketoacids α-ketobutyrate and pyruvate over the natural branched substrate α-ketoisovalerate.     

Cation induced differential effect on structural and functional properties of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> α-Isopropylmalate synthase

Background  

α-isopropylmalate synthase (MtαIPMS), an enzyme that catalyzes the first committed step of the leucine biosynthetic pathway of Mycobacterium tuberculosis is a potential drug target for the anti-tuberculosis drugs. Cations induce differential effect of activation and inhibition of MtαIPMS. To date no concrete mechanism for such an opposite effect of similarly charged cations on the functional activity of enzyme has been presented.     

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

α-Isopropylmalate synthase (α-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 α-IPMS proteins from other bacteria. NmeIPMS is a homodimer which catalyses the condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA), and is inhibited by leucine. NmeIPMS can use alternate α-ketoacids as substrates and, in contrast to α-IPMS from other sources, is activated by a range of metal ions including Cd²⁺ and Zn²⁺ that have previously been reported as inhibitory, since they suppress the dithiodipyridone assay system rather than the enzyme itself. Previous studies indicate that α-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 α-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.     

In vitro reactions of isopropyl methanesulfonate with DNA and with 2'-deoxyribonucleosides

Isopropyl methanesulfonate (IPMS), an SN1 alkylating agent, is a direct-acting mutagen in bacteria. We recently reported that s.c. and topical administration of IPMS to mice resulted in the rapid induction of thymic lymphomas. Thymic lymphoma induction was not observed following administration of the SN2 alkylating agents methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). We have studied the reactions of IPMS with dAdo, dCyd, dGuo and dThd at pH 6.5 to 7.5 and 37 degrees C for 3 h. IPMS formed the following isopropyl (IP) adducts: 7-IP-Gua (4% yield), O6-IP-Gua (8%), O2-IP-Cyt (1%), O2-IP-dThd (2%), 3-IP-dThd (1%), and O4-IP-dThd (0.4%). Adducts were characterized from UV and mass spectra. IPMS was reacted in vitro with calf thymus DNA (pH 6.5 to 7.5, 37 degrees C, 3 h) and yielded (nmol/mg DNA): 7-IP-Gua (22) O6-IP-dGuo (11), O2-IP-Cyt (9), O2-IP-dThd (2), O4-IP-dThd (2), 3-IP-Ade (0.2) and 3-IP-dThd (0.2). The relatively greater alkylation of exocyclic oxygen atoms in DNA by IPMS compared to values for MMS and EMS reported by others, may play a role in the induction of thymic lymphomas in mice by IPMS and the lack of such activity by MMS and EMS.     

Expression of a Brassica Isopropylmalate Synthase Gene in Arabidopsis Perturbs Both Glucosinolate and Amino Acid Metabolism

Isopropylmalate synthase (IPMS) is a key enzyme in the biosynthesis of the essential amino acid leucine, and thus primary metabolism. In Arabidopsis, the functionally similar enzyme, methythiolalkylmalate synthase (MAM), is an important enzyme in the elongation of methionine prior to glucosinolate (GSL) biosynthesis, as part of secondary metabolism. We describe the cloning of an IPMS gene from Brassica, BatIMS, and its functional characterisation by heterologous expression in E. coli and Arabidopsis. Over expression of BatIMS in Arabidopsis resulted in plants with an aberrant phenotype, reminiscent of mutants in GSL biosynthesis. Metabolite analyses showed that these plants had both perturbed amino acid metabolism and enhanced levels of GSLs. Microarray profiling showed that BatIMS over expression caused up regulation of the genes for methionine-derived GSL biosynthesis, and down regulation of genes involved in leucine catabolism, in addition to perturbed expression of genes involved in auxin and ethylene metabolism. The results illustrate the cross talk that can occur between primary and secondary metabolism within transgenic plants. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

From amino acid to glucosinolate biosynthesis: protein sequence changes in the evolution of methylthioalkylmalate synthase in Arabidopsis

Methylthioalkylmalate synthase (MAM) catalyzes the committed step in the side chain elongation of Met, yielding important precursors for glucosinolate biosynthesis in Arabidopsis thaliana and other Brassicaceae species. MAM is believed to have evolved from isopropylmalate synthase (IPMS), an enzyme involved in Leu biosynthesis, based on phylogenetic analyses and an overlap of catalytic abilities. Here, we investigated the changes in protein structure that have occurred during the recruitment of IPMS from amino acid to glucosinolate metabolism. The major sequence difference between IPMS and MAM is the absence of 120 amino acids at the C-terminal end of MAM that constitute a regulatory domain for Leu-mediated feedback inhibition. Truncation of this domain in Arabidopsis IPMS2 results in loss of Leu feedback inhibition and quaternary structure, two features common to MAM enzymes, plus an 8.4-fold increase in the k(cat)/K(m) for a MAM substrate. Additional exchange of two amino acids in the active site resulted in a MAM-like enzyme that had little residual IPMS activity. Hence, combination of the loss of the regulatory domain and a few additional amino acid exchanges can explain the evolution of MAM from IPMS during its recruitment from primary to secondary metabolism.     

Biosynthesis of Branched-Chain Amino Acids in Yeast: Correlation of Biochemical Blocks and Genetic Lesions in Leucine Auxotrophs

The three enzymatic steps in the conversion of alpha-ketoisovalerate to alpha-ketoisocaproate were examined in wild-type and in leucine auxotrophic stocks of yeast. Procedures for the reliable assay of each of the enzymatic steps in crude extracts were devised. Crude extracts of the prototrophic haploid stock catalyzed all three enzymatic steps. Examination of a series of leucine auxotrophs permitted a correlation between the three enzymatic steps and the genetic lesions affecting 10 different loci. This examination revealed that a single locus (le-6) affected primarily alpha-isopropylmalate synthetase, the first step in the pathway. Lesions in six loci (le-1, le-4, le-5, le-7, le-8, and le-10) lead primarily to a deficiency in the activity of the second enzyme in the pathway, alpha-isopropylmalate isomerase. Stocks with lesions in three loci (le-2, le-3, and le-9) were primarily blocked in the third step of the pathway, catalyzed by beta-isopropylmalate dehydrogenase. The results with the mutants provide strong evidence that the pathway for leucine biosynthesis proposed by Strassman and his colleagues is the sole significant pathway in yeast.     

Branched chain amino acid regulation of the ILV2 locus in Saccharomyces cerevisiae

Mutant regulatory loci of the branched pathway for the biosynthesis of isoleucine-valine and leucine were identified with the unusual phenotype of an amino acid dependent auxotrophy. Two mutant loci, bcs1 and bcs2, conferred branched chain amino acid sensitivity and showed independent segregation. Linkage studies defined bcs1 as a cis-acting regulatory site of ILV2 (SMR1). ILV2 upstream deletion analyses and high-copy transformation of the positive regulatory locus LEU3 ruled out the possibility of LEU3 protein binding palindromes mediating the branched chain amino acid dependent auxotrophy. In the presence of leucine and valine, the general amino acid control system (GCN4) was epistatic to bcs1 and bcs2, and under nonstarvation conditions GCN4 strains showed an increased acetolactate synthase activity over gcn4 strains. Thus in addition to general regulation of ILV2, GCN4 functions in basal level expression when the locus is subject to specific repression by pathway end product.     

A common F-box gene regulates the leucine homeostasis of Medicago truncatula and Arabidopsis thaliana

The F-box domain is a conserved structural protein motif that most frequently interacts with the SKP1 protein, the core of the SCFs (SKP1-CULLIN-F-box protein ligase) E3 ubiquitin protein ligases. As part of the SCF complexes, the various F-box proteins recruit substrates for degradation through ubiquitination. In this study, we functionally characterized an F-box gene (MtF-box) identified earlier in a population of Tnt1 retrotransposon-tagged mutants of Medicago truncatula and its Arabidopsis thaliana homolog (AtF-box) using gain- and loss-of-function plants. We highlighted the importance of MtF-box in leaf development of M. truncatula. Protein–protein interaction analyses revealed the 2-isopropylmalate synthase (IPMS) protein as a common interactor partner of MtF-box and AtF-box, being a key enzyme in the biosynthesis pathway of the branched-chain amino acid leucine. For further detailed analysis, we focused on AtF-box and its role during the cell division cycle. Based on this work, we suggest a mechanism for the role of the studied F-box gene in regulation of leucine homeostasis, which is important for growth.

     

The response of internal dynamics to hydrophobic core mutations in the SH3 domain from the Fyn tyrosine kinase

We have used (15)N- and (2)H-NMR spin relaxation experiments to study the response of backbone and side-chain dynamics when a leucine or valine is substituted for a completely buried phenylalanine residue in the SH3 domain from the Fyn tyrosine kinase. Several residues show differences in the time scales and temperature dependences of internal motions when data for the three proteins are compared. Changes were also observed in the magnitude of dynamics, with the valine, and to a lesser extent leucine mutant, showing enhanced flexibility compared to the wild-type (WT) protein. The motions of many of the same amide and methyl groups are affected by both mutations, identifying a set of loci where dynamics are sensitive to interactions involving the targeted side chain. These results show that contacts within the hydrophobic core affect many aspects of internal mobility throughout the Fyn SH3 domain.     

The spectrum of intermediate syndrome following acute organophosphate poisoning: a prospective cohort study from Sri Lanka

Background

Intermediate syndrome (IMS) is a major cause of death from respiratory failure following acute organophosphate poisoning. The objective of this study was to determine repetitive nerve stimulation (RNS) predictors of IMS that would assist in patient management and clinical research.

Methods and Findings

Seventy-eight consenting symptomatic patients with organophosphate poisoning were assessed prospectively with daily physical examination and RNS. RNS was done on the right and left median and ulnar nerves at 1, 3, 10, 15, 20, and 30 Hz. The study was conducted as a prospective observational cohort study in the Central Province, Sri Lanka. IMS was diagnosed in ten out of 78 patients using a priori clinical diagnostic criteria, and five of them developed respiratory failure. All ten patients showed progressive RNS changes correlating with the severity of IMS. A decrement-increment was observed at intermediate and high frequencies preceding the onset of clinical signs of IMS. As the patient developed clinical signs of IMS, decrement-increment was progressively noted at low and intermediate frequencies and a combination of decrement-increment and repetitive fade or severe decrement was noted at high frequencies. Severe decrement preceded respiratory failure in four patients. Thirty patients developed forme fruste IMS with less severe weakness not progressing to respiratory failure whose RNS was characterized by decrement-increment or a combination of decrement-increment and repetitive fade but never severe decrements.

Conclusions

Characteristic changes in RNS, preceding the development of IMS, help to identify a subgroup of patients at high risk of developing respiratory failure. The forme fruste IMS with the characteristic early changes on RNS indicates that IMS is a spectrum disorder. RNS changes are objective and precede the diagnosis and complications of IMS. Thus they may be useful in clinical management and research.     

Evolutionary genetics of Metridium senile. II. Geographic patterns of allozyme variation

Electrophoretic surveys have demonstrated that populations of the sea anemone Metridium senile along the northeast coast of the United States are polymorphic at four enzyme loci. Phosphoglucose isomerase (PGI) has two alleles in most populations, phosphoglucomutase (PGM) has three alleles, and two leucine aminopeptidase loci have two common alleles each. Phosphoglucose isomerase displays clinal variation and an apparent association with environmental temperature. Phosphoglucomutase shows clinal variation north of Cape Cod for two of the three alleles, while the two leucine aminopeptidase loci are not clinal. All loci show a great deal of variation in populations on Cape Cod, but there is no apparent systematic pattern to this variation. Temperature may be a selective agent in the maintenance of the PGI and PGM clines, although other possibilities cannot presently be completely excluded.Supported by Grant T-4 from the Health Research and Services Foundation, NSF DEB77-14442, NIH GM25809, and NIH GM28024.     

Immunomagnetic separation of Toxoplasma gondii oocysts using a monoclonal antibody directed against the oocyst wall

Recent outbreaks of waterborne toxoplasmosis have stimulated the development of sensitive methods to detect Toxoplasma gondii oocysts in samples suspected to be contaminated. The immunomagnetic separation (IMS) have been standardised to detect waterborne protozoa, but it did not exist for Toxoplasma oocysts. In this study, we describe two monoclonal antibodies (mAbs 3G4 and 4B6) produced against the oocyst wall, and the incorporation of mAb 3G4 in an IMS procedure. We found that an indirect IMS method gave better mean recoveries than a direct one (69.4% and 25.2%, respectively). Dissociation of oocyst_magnetic bead complexes was greatly improved by using a 2% aqueous H2SO4 solution instead of a 0.1 N HCl solution (82.8% and 17.4%, respectively). With these parameters, mean recoveries of less than 1000 oocysts ranged from 44.6% to 82.9%, depending on incubating temperature and buffer. Age of oocysts (1 or 12 months old) does not influence IMS performances. Results of this study indicate that the described IMS is an efficient technique to recover Toxoplasma oocysts.     

Salmonella Locus Affecting Phosphoenolpyruvate Synthase Activity Identified by a Deletion Analysis

Strain leu-4017, derived from Salmonella typhimurium LT2, cannot utilize acetate, pyruvate, or citric acid cycle intermediates as sole sources of carbon. The mutation in this strain extends from the A cistron of the leucine operon to some point between leu and azi, presumably deleting one or more loci involved in the utilization of these compounds. One of these loci is required for phosphoenolpyruvate synthase activity.     

The essential mitochondrial protein Erv1 cooperates with Mia40 in biogenesis of intermembrane space proteins

The proteins of the mitochondrial intermembrane space (IMS) are encoded by nuclear genes and synthesized on cytosolic ribosomes. While some IMS proteins are imported by the classical presequence pathway that involves the membrane potential deltapsi across the inner mitochondrial membrane and proteolytic processing to release the mature protein to the IMS, the import of numerous small IMS proteins is independent of a deltapsi and does not include proteolytic processing. The biogenesis of small IMS proteins requires an essential mitochondrial IMS import and assembly protein, termed Mia40. Here, we show that Erv1, a further essential IMS protein that has been reported to function as a sulfhydryl oxidase and participate in biogenesis of Fe/S proteins, is also required for the biogenesis of small IMS proteins. We generated a temperature-sensitive yeast mutant of Erv1 and observed a strong reduction of the levels of small IMS proteins upon shift of the cells to non-permissive temperature. Isolated erv1-2 mitochondria were selectively impaired in import of small IMS proteins while protein import pathways to other mitochondrial subcompartments were not affected. Small IMS precursor proteins remained associated with Mia40 in erv1-2 mitochondria and were not assembled into mature oligomeric complexes. Moreover, Erv1 associated with Mia40 in a reductant-sensitive manner. We conclude that two essential proteins, Mia40 and Erv1, cooperate in the assembly pathway of small proteins of the mitochondrial IMS.     

Hereditary multiple exostoses (EXT): mutational studies of familial EXT1 cases and EXT-associated malignancies.

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the formation of cartilage-capped prominences that develop from the growth centers of the long bones. EXT is genetically heterogeneous, with three loci, currently identified on chromosomes 8q24.1, 11p13, and 19q. The EXT1 gene, located on chromosome 8q24.1, has been cloned and is encoded by a 3.4-kb cDNA. Five mutations in the EXT1 gene have been identified--four germ-line mutations, including two unrelated families with the same mutation, and one somatic mutation in a patient with chondrosarcoma. Four of the mutations identified resulted in frameshifts and premature termination codons, while the fifth mutation resulted in a substitution of leucine for arginine. Loss of heterozygosity (LOH) analysis of chondrosarcomas and chondroblastomas revealed multiple LOH events at loci on chromosomes 3q, 8q, 10q, and 19q. One sporadic chondrosarcoma demonstrated LOH for EXT1 and EXT3, while a second underwent LOH for EXT2 and chromosome 10. A third chondrosarcoma underwent LOH for EXT1 and chromosome 3q. These results agree with previous findings that mutations at EXT1 and multiple genetic events that include LOH at other loci may be required for the development of chondrosarcoma.