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PubMed2002

An isoleucine-leucine substitution in chloroplastic acetyl-CoA carboxylase from green foxtail (Setaria viridis L. Beauv.) is responsible for resistance to the cyclohexanedione herbicide sethoxydim.

The cDNAs encoding chloroplastic acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) from three lines of Setaria viridis (L. Beauv.) resistant or sensitive to sethoxydim, and from one sethoxydim-sensitive line of Setaria italica (L. Beauv.) were cloned and sequenced. Sequence comparison revealed that a single isoleucine-leucine substitution discriminated ACCases from sensitive and resistant lines. Using near-isogenic lines of S. italica derived from interspecific hybridisation, we demonstrated that the transfer of the S. viridis mutant ACCase allele into a sethoxydim-sensitive S. italica line conferred resistance to this herbicide. We confirmed this result using allele-specific polymerase chain reaction and showed that a single copy of the mutant allele is sufficient to confer resistance to sethoxydim. We conclude that a mutant allele of chloroplastic ACCase encoding a leucine residue instead of an isoleucine residue at position 1780 is a major gene of resistance to sethoxydim.

Acetyl-CoA Carboxylase↗
PubMed1985

Properties of a microsomal enzyme system from Linum usitatissimum (linen flax) which oxidizes valine to acetone cyanohydrin and isoleucine to 2-methylbutanone cyanohydrin.

Microsomal preparations from flax seedlings have recently been shown to convert L-valine to acetone cyanohydrin, the precursor of the cyanogenic glucoside linamarin [A. J. Cutler and E. E. Conn (1981) Arch. Biochem. Biophys. 212, 468-474]. Further details of this four-step biosynthetic sequence and also details of the analogous reactions in lotaustralin biosynthesis have been obtained. The lotaustralin precursor, 2-methylbutyraldoxime, is the best substrate for cyanide production (Vmax = 413 nmol h-1 g fresh wt-1) and inhibits the conversion of valine and isoleucine into products. Similarly, the linamarin precursor isobutyraldoxime is an excellent substrate (Vmax = 400 nmol h-1 g fresh wt-1) and also inhibits oxidation of the amino acids. The substrate specificity of the oxime-metabolizing step is low and a variety of aliphatic oximes are converted to cyanide. On the other hand, the activity of the microsomal extract is highly selective with regard to the amino acid substrate since, of the aliphatic amino acids tested, only valine and isoleucine are metabolized. We were unable to demonstrate product formation from isobutyronitrile (a linamarin precursor) but did observe detectable cyanide formation from 2-methylcyanobutane, the corresponding precursor of lotaustralin. Competition experiments showed that the biosynthesis of linamarin and lotaustralin is not likely to be catalyzed by separate enzyme systems.

Chromatography, Gas↗
PubMed1986

Amino acid catabolism by perfused rat hindquarters: degradation of threonine and isoleucine.

Rat hindquarters were perfused without added substrate other than trace amounts of [U-14C]threonine or [U-14C]isoleucine. Comparison of incorporation of radiolabel into some nonessential amino acids, citrate cycle intermediates, and lactate is presented. Activities of three enzymes for the initial reactions in threonine degradation are reported. It is concluded that skeletal muscle catabolizes threonine, and that the latter is a potential source of carbon for glucogenic precursors for the liver. In contrast, label from isoleucine was incorporated into glutamate, glutamine, and alanine much more than was that from threonine. Large amounts of organic acids accumulated, and more than 60% of total radioactivity was lost as CO2 during a 2-h perfusion period.

Amino Acids↗
PubMed1989

Inactivation of human arginine-143, lysine-143, and isoleucine-143 Cu,Zn superoxide dismutases by hydrogen peroxide: multiple mechanisms for inactivation.

Site-specific mutants of human Cu,Zn superoxide dismutase (Cu,ZnSOD) have been prepared in which the active-site arginine at position 143 (i.e., SODR143) has been replaced by either lysine (SODK143) or isoleucine (SODI143). As reported previously (W.F. Beyer, Jr., et al. (1987) J. Biol. Chem. 262, 11182-11187), SODK143 and SODI143 have 43 and 11%, respectively, of the catalytic activity of SODR143. H2O2, at low concentrations, acts as an affinity reagent for the inactivation of SODR143. At pH 9.0 and 25 degrees C, the process is characterized by a half-saturation constant for H2O2, K50, of 5.1 mM and a maximum pseudo-first-order rate constant for inactivation, Kmax, of 0.53 min-1. At pH 11.5, the corresponding values are 0.63 mM and 1.23 min-1. The active species in the inactivation is likely HO2-, as previously found with yeast and bovine Cu,ZnSODs (see C.L. Borders, Jr., and I. Fridovich (1985) Arch. Biochem. Biophys. 241, 472-476). SODK143 is also inactivated by HO2- by an affinity mechanism, i.e., one where reversible binding of H2O2 (HO2-) is a prerequisite for inactivation. At pH values of 9.0 and 11.5, the kmax values are 0.92 and 1.08 min-1, respectively; however, the corresponding K50 values increase to 42.5 and 15.8 mM, respectively. SODI143 is also inactivated by H2O2, but no evidence for an affinity mechanism was found; instead, a second-order kinetic mechanism was observed. Inactivation of each of the three enzymes is accompanied by the loss of one histidine per subunit. At elevated concentrations of H2O2, a second nonaffinity mechanism of inactivation of both SODR143 and SODK143 was found, in which a second equivalent of H2O2 reacts with the Cu,ZnSOD.HO2- complex to give a competing second-order inactivation. It appears that the positive charge of arginine-143 plays a role in the binding of HO2- at the active site of human Cu,ZnSOD, and that replacement of the arginine by lysine gives an enzyme with a similar affinity mechanism of inactivation, but with a greatly reduced affinity for HO2-. However, replacement with isoleucine causes an entirely different mechanism of inactivation; this raises the possibility that the mechanism of enzyme catalysis of superoxide dismutation by SODI143 is also different.

Arginine↗
PubMed1977

Partial derepression of the isoleucine-valine enzymes during methionine starvation is Salmonella typhimurium.

Methionine starvation of methionine auxotrophs in the presence of excess branched-chain amino acids results in a partial derepression of the isoleucine and valine enzymes. Reversed-phase chromatography indicated that isoleucine, valine and leucine tRNA were altered during methionine starvation. In addition, the total tRNA isolated from cells under these conditions were undermethylated. The observed derepression may be caused by the inability of methyl-deficient tRNA's to participate adequately in normal regulatory functions.

2-Isopropylmalate Synthase↗
PubMed1995

Identification of a clinical isolate of HIV-1 with an isoleucine at position 82 of the protease which retains susceptibility to protease inhibitors.

The HIV-1 protease (PR) is essential for the production of mature virions. As such, it has become a target for the development of anti-HIV chemotherapeutics. Multiple passages of virus in cell culture in the presence of PR inhibitors have resulted in the selection of variants with decreased sensitivity to inhibitors of the PR. The most common alteration observed is a single amino acid change at position 82. This particular position has been well characterized by several laboratories as being important for the susceptibility of the virus to inhibitors of PR function. Mutations which result in the substitution of the wild-type valine with alanine, phenylalanine, threonine or isoleucine at position 82 of the PR have been associated with decreased sensitivity to several PR inhibitors. We describe here a clinical strain of HIV-1 that contains an isoleucine at position 82 of the PR instead of the usual valine. This strain is unique in that it was isolated from a patient that was anti-retroviral naive, and in the past, variants at position 82 of the PR have only been found after treatment of patients or cell culture with PR inhibitors. Moreover, this virus remains sensitive to PR inhibitors of the cyclic urea and C-2 symmetrical diol classes.

Amino Acid Sequence↗
PubMed2006

How to discriminate between leucine and isoleucine by low energy ESI-TRAP MSn.

In peptide sequencing experiments involving a single step tandem mass acquisition, leucine and isoleucine are indistinguishable because both are characterized by a 113 Da mass difference from the other peptide fragments in the MS2 spectrum. In this work, we propose a new method to distinguish between these two amino acids in consecutive MSn experiments, exploiting a gas-phase fragmentation of isoleucine that leads to a diagnostic 69 Da ion. We used this method to assess the Leu/Ile residues of several synthetic peptides. The procedure was then tested on a tryptic digest of myoglobin, assigning the correct amino acid in the majority of the peptides. This work was performed with an old and low-resolution instrument, thus demonstrating that our method is suitable for a wide number of ion trap mass spectrometers, not necessarily expensive or up-to-date.

Amino Acid Sequence↗
PubMed2006

Inborn errors of isoleucine degradation: a review.

Three inborn errors have been identified in the pathway of isoleucine degradation. Deficiency of beta-ketothiolase (beta-KT, also known as T2, mitochondrial acetoacetyl-CoA thiolase and acetyl-CoA acetyltransferase 1) is a well-described disorder which presents with acute episodic ketoacidosis. In contrast, short/branched-chain acyl-CoA dehydrogenase (SBCAD) and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiencies are recently described and relatively rare defects which present with predominantly neurological manifestations, although acute metabolic decompensation may occur in the early newborn period. Careful examination of urine organic acids is required for identification and differential diagnosis of these disorders, with awareness that the abnormalities may be subtle and variable. Tandem MS analysis of acylcarnitines may reveal elevated C5 (SBCAD) or C5:1 and/or OH-C5 species (MHBD and beta-KT deficiencies) but the abnormalities are non-diagnostic and may be intermittent or absent. Confirmation of diagnosis is therefore advisable by specific enzyme assay and/or mutation analysis of the ACAT1 (beta-KT), ACADSB (SBCAD) or HADH2 (MHBD) genes. The latter is located on the X chromosome, accounting for the milder clinical phenotype in females. If beta-KT deficiency is diagnosed early and treated by fasting avoidance and modest protein restriction, ketoacidosis episodes can be prevented and the prognosis is excellent. The role of treatment in SBCAD deficiency remains unclear pending further delineation of its clinical phenotype and pathogenicity, particularly regarding asymptomatic individuals detected by expanded newborn screening. The ineffectiveness of isoleucine restriction in MHBD deficiency is consistent with the additional roles of this multifunctional enzyme in sex steroid and neurosteroid metabolism and its interaction with amyloid-beta peptide.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2004

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency: impaired catabolism of isoleucine presenting as neurodegenerative disease.

We describe a further case of recently reported 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency, a disorder of isoleucine metabolism. The development of pronounced brain atrophy and symmetrical alterations of the basal ganglia were observed and the importance of specific enzymatic tests is emphasized, which should be performed if urinary metabolites suggest impaired catabolism of isoleucine.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1991

Increased collagen gene expression in vascular smooth muscle cells cultured in serum or isoleucine deprived medium.

The relationship between type I and type III collagen gene expression and the growth state of cultured bovine vascular smooth muscle cells has been investigated. Growth was modulated by incubation of subconfluent cultures of smooth muscle cells in media containing concentrations of fetal calf serum ranging from 10% to 0.5%. As the serum concentration was lowered, the doubling time increased from 17 hours to 70 hours, and collagen mRNA levels increased as judged by Northern blot analysis. The levels of induction were 5- to 15-fold for alpha 1(I), 2- to 3-fold for alpha 2(I), and 4- to 7-fold for alpha 1(III). Metabolic cell labelling with precursor amino acids indicated that type I and type III procollagen synthesis was elevated approximately 2-fold as growth slowed. A similar 2-fold increase occurred upon translation of isolated RNAs in a rabbit reticulocyte cell-free system. Nuclear run-on analysis indicated that increased collagen transcription can account for part of the increase in mRNA levels following incubation in lowered fetal calf serum. To further examine the influence of proliferation on collagen gene expression, smooth muscle cells were cultured in isoleucine-free medium, which resulted in quiescence within 36 hours. Induction of collagen mRNA levels was observed within 24 hours of incubation in isoleucine-free medium. Thus, collagen gene expression increases as the growth of vascular smooth muscle cells is slowed by either deprivation of growth factors or essential amino acids. Regulation is mediated at several sites, including gene transcription and mRNA translation, and possibly post-transcriptional steps.

Animals↗
PubMed1980

Primary structure of AUA-specific isoleucine transfer ribonucleic acid from Escherichia coli.

The nucleotide sequence of an E. coli isoleucine tRNA (tRNAIle minor) specific for the codon AUA was determined by postlabeling procedures using only 2.5 micrograms (0.05 A260 unit) of the material. The sequence was pG-G-C-C-C-C-U-s4U-A-G-C-U-C-A-G-U-Gm-G-D-D-A-G-A-G-C-A-A-G-C-G-A-C-U-N+-A-U-t6A-A-psi-C-G-C-U-U-G-m7G-acp3U-C-G-C-U-G-G-T-psi-C-A-A-G-U-C-C-A-G-C-A-G-G-G-G-C-C-A-C-C-AOH. The nucleotide sequences in the regions of the D arm and T psi C arm of tRNAIle minor were quite similar to the corresponding regions of tRNAIle major. However, the sequences in the CCA stem and anticodon stem of tRNAIle minor were different from those of tRNAIle major. The overall homology between the two isoleucine tRNAs was 68%. E. coli tRNALys, tRNAMet, tRNAValIIA and tRNAArg also have relatively high sequence homology with tRNAIle minor.

Base Sequence↗
PubMed1996

An isoleucine to leucine mutation that switches the cofactor requirement of the EcoRV restriction endonuclease from magnesium to manganese.

The EcoRV restriction endonuclease cleaves DNA at its recognition sequence more readily with Mg2+ as the cofactor than with Mn2+ but, at noncognate sequences that differ from the EcoRV site by one base pair, Mn2+ gives higher rates than Mg2+. A mutant of EcoRV, in which an isoleucine near the active site was replaced by leucine, showed the opposite behavior. It had low activity with Mg2+, but, in the presence of Mn2+ ions, it cleaved the recognition site faster than wild-type EcoRV with either Mn2+ or Mg2+. The mutant was also more specific for the recognition sequence than the native enzyme: the noncognate DNA cleavages by wild-type EcoRV and Mn2+ were not detected with the mutant. Further mutagenesis showed that the protein required the same acidic residues at its active site as wild-type EcoRV. The Ile-->Leu mutation seems to perturb the configuration of the metal-binding ligands at the active site so that the protein has virtually no affinity for Mg2+ yet it can still bind Mn2+ ions, though the latter only occurs when the protein is at the recognition site. This contrasts to wild-type EcoRV, where Mn2+ ions bind readily to complexes with either cognate and noncognate DNA and only Mg2+ shows the discrimination between the complexes. The structural perturbation is a specific consequence of leucine in place of isoleucine, since mutants with valine or alanine were similar to wild-type EcoRV.

Base Sequence↗
PubMed2005

Coded amino acids in gas phase: the shape of isoleucine.

The solid alpha-amino acid isoleucine has been vaporized by laser ablation and expanded in a supersonic jet, where the molecular conformations of the isolated molecule were probed using Fourier transform microwave spectroscopy. Two conformers of neutral isoleucine have been detected in gas phase, the most stable being stabilized by an intramolecular hydrogen bond N-H...O=C and a cis-COOH arrangement. The higher energy form is stabilized by an intramolecular hydrogen bond N...H-O. The sec-butyl side chain of the amino acid adopts the same configuration in the two observed conformers, with a staggered configuration at Cbeta similar to that observed in valine and a trans arrangement of Calpha and Cdelta. Ab initio calculations at MP2/6-311++G(d,p) level reproduce satisfactorily the experimental results.

Hydrogen Bonding↗
PubMed2004

Mutation of a highly conserved isoleucine disrupts hydrophobic interactions in the alpha beta spectrin self-association binding site.

We studied an infant with severe neonatal hemolytic anemia and hyperbilirubinemia that evolved into a partially compensated ellipto-poikilocytic anemia. His father had typical elliptocytosis. Their erythrocyte membranes demonstrated structural and functional defects in spectrin. Genetic studies revealed that the proband and his father were heterozygous for an alpha-spectrin mutation, Ile24Thr, in the alpha beta spectrin self-association binding site. The proband also carried the low expression allele alpha(LELY) in trans, influencing the clinical phenotype. The importance of isoleucine in this position of the proposed triple helical model of spectrin repeats is highlighted by its evolutionary conservation in all alpha spectrins from Drosophila to humans. Molecular modeling demonstrated that replacement of a hydrophobic isoleucine with a hydrophilic threonine disrupts highly conserved hydrophobic interactions in the interior of the spectrin triple helix critical for spectrin function.

Binding Sites↗
PubMed1991

Methylmalonic acid metabolism and nervous-system fatty acids in cobalamin-deficient fruit bats receiving supplements of methionine, valine and isoleucine.

Cobalamin neuropathy was produced in Cape fruit bats (Rousettus aegyptiacus) by a cobalamin-free diet combined with intermittent exposure to nitrous oxide, which inactivates cobalamin. Cobalamin-deficient bats had low hepatic methylmalonyl-CoA mutase holoenzyme activity, with elevated plasma and urinary methylmalonic acid levels. No significant changes could be demonstrated in the concentration of odd- or branched-chain fatty acids in the nervous system. Supplementation of the cobalamin-free diet with methionine, valine or isoleucine delayed the onset of neuropathy, despite persistence of methylmalonic acid accumulation. Supplementation with any of the three amino acids was associated with elevation of hepatic methionine concentration. The action of valine and isoleucine in delaying the onset of neuropathy can be explained by their methionine-sparing effect. These results emphasize the central role of methionine in the cobalamin neuropathy and do not support the hypothesis that the neuropathy is related to an effect of cobalamin deficiency on the propionic acid pathway.

Amino Acids↗
PubMed1996

Isoleucine 368 is involved in low-affinity binding of N6-modified cAMP analogues to site B of the regulatory subunit of cAMP-dependent protein kinase I.

The regulatory (R) subunit of cAMP-dependent protein kinase has a well-defined domain structure including the two in-tandem cAMP-binding sites that constitute the C-terminus of the protein. The N-terminal binding site (A) has a considerably higher affinity for analogues of cAMP that are substituted with bulky and hydrophobic substituents at the 6-amino group of the adenine ring compared to the affinity observed at the second site (B). On the basis of the crystal structure of the catabolite gene activator protein from Escherichia coli, molecular modelling of the binding domains suggested that a tyrosine (Y244) in site A could be involved in a high-affinity hydrophobic interaction, whereas a corresponding isoleucine (I368) in domain B could lead to steric hindrance in the binding of bulky N6-substituted analogues. Site-directed mutagenesis was used to construct mutations in Y244 and I368. Binding displacement experiments showed that replacing the tyrosine in site A with isoleucine (Y244I) did not affect the interaction of either N6-substituted or otherwise modified analogues with this site. However, replacing I368 with tyrosine (I368Y) led to a 3-4-fold increase in affinity for those N6-modified analogues that had a hydrophobic group attached directly or close to the 6-amino molecule. We conclude that I368 is involved in the molecular interaction between binding domain B and the 6-amino group of the adenine moiety of cAMP and that this residue is partly responsible for the reduced affinity of N6-substituted cAMP analogues for this site.

Amino Acid Sequence↗
PubMed1974

Pleiotropy of hisT mutants blocked in pseudouridine synthesis in tRNA: leucine and isoleucine-valine operons.

The hisT gene codes for an enzyme responsible for the conversion of uridine to pseudouridine (Psi) in the anticodon region of many tRNA species in Salmonella typhimurium. We have previously shown that a hisT mutant has tRNA(His) which lacks pseudouridine in this region and as a consequence has an altered chromatographic behavior. We show here a similar alteration in chromatographic behavior of all tRNA(Leu) and one tRNA(Ile) species from a hisT mutant. By contrast, tRNA(Val), which contains no pseudouridine except for the one in the TPsiCG sequence, is chromatographically unaltered in a hisT mutant. The absence of pseudouridine in the anticodon region of tRNA in hisT mutants has been previously shown to cause derepression of the histidine operon. We show here that in hisT mutants the regulation of the leucine and the isoleucine and valine operons is also affected: the enzymes of these operons are refractory to repression by the branched chain amino acids. However, there is no difference between hisT and wild type in the pattern of derepression caused by isoleucine or valine limitation and only a slight difference in the enzyme levels in cells grown on minimal medium. The alteration in the regulation of branched chain amino acid operons may also explain why hisT mutants are resistant to inhibition of growth by the amino acid analogues 5,5,5-trifluoroleucine, beta-hydroxyleucine, and norleucine and by the oligopeptides glycylglycylnorleucine and norleucylnorleucine.

Acids↗
PubMed1975

Amino acid requirements of the young turkey: leucine, isoleucine and valine.

1. The leucine, isoleucine and valine requirements of the 3-week-old turkey were determined in a factorial experiment and were found to be 1-42, 0-84 and 1-21% respectively in semi-purified diets containing 12-5 MJ ME/kg. 2. It is suggested that the efficiency with which turkeys and chicks utilise isoleucine and valine is similar, and that a daily intake of about 325 mg of valine would sustain a rate of growth of about 23 g/d in both species.

Animals↗