The ilvC gene product, acetohydroxy acid isomeroreductase, an enzyme essential for isoleucine and valine formation, is subject to substrate induction in Escherichia coli. We have isolated a mutant of E. coli K-12 with a mutation that renders the ilvC gene product noninducible by its substrates, the acetohydroxy acids. This mutation, ilvY466, has been shown to be in a previously undiscovered locus that lies between ilvC and ilvO. The ilvY product, upsilon, is thought to be a regulatory element involved in the induction of ilvC. We postulate the recognition site, ilvQ, or upsilon and suggest that it lies between ilvC and ilvB. A possible model, involving upsilon, in the positive control of isomeroreductase is presented. Pleiotropic effects of the ilvY466 mutation have been recognized from changes in the end-product inhibition of threonine deaminase and of acetohydroxy acid synthetase. In addition, pleiotropic effects of this lesion on the regulation of threonine deaminase and the physical properties of threonine deaminase and acetohydroxy acid synthetase have been observed.
Thiaisoleucine-resistant mutants of Escherichia coli strain K-12 which exhibited reduced isoleucyl soluble ribonucleic acid synthetase activity were isolated. Resistance was apparently achieved by the selection of a synthetase with a 10-fold decrease in apparent affinity for thiaisoleucine. This mutation also resulted in a 2.5-fold decrease in apparent affinity for the natural substrate, l-isoleucine, and less activity than found in wild type. The mutants grew more slowly than wild type and were derepressed for three of the five enzymes in the pathways to isoleucine and valine.
The levels of the five enzymes required for isoleucine and valine synthesis were examined under several growth conditions in strain K-12 of Escherichia coli and mutants derived from it. In strains with wild type repressibility, the same pattern of derepression was found on limiting isoleucine as is found to be constitutive in strain Tir-8, which has an altered isoleucine-activating enzyme. Homoserine dehydrogenase, which is essential for the biosynthesis of threonine and is normally derepressed on limiting isoleucine or threonine, is also derepressed in strain Tir-8. Threonine deaminase and homoserine dehydrogenase were partially repressed in strain Tir-8 by very high levels of isoleucine, but were not further derepressed over levels in minimal medium by limiting isoleucine.
An abnormal hemoglobin, termed Hb Savannah, was found in red cell hemolysate of a young Caucasian girl with severe hemolytic anemia. The presence of this unstable variant became evident when inclusion bodies appeared rapidly upon exposure of red cells to redox dyes and a large percentage of hemoglobin in hemolysate precipitated on warming to 65°C. Treatment of the hemoglobin with p-hydroxymercuribenzoate (PMB) caused a rapid dissociation into monomers; starch-gel electrophoresis of PMB-treated hemoglobin showed the presence of abnormal β-chains. Data from structural studies of isolated β-chains indicated substitution of a valyl residue for the normally occurring glycyl residue at position 24, which corresponds to helical residue B6. A similar substitution but with an arginine replacing the glycyl residue has been observed in Hb Riverdale-Bronx. The glycine to valine substitution will change the relationship of the B and the E helices which results in extensive conformational changes in the β-chain. This change presumably causes an increased dissociation of the hemoglobin molecule into dimers and probably monomers, and a decreased stability of the αβ-dimers. The hemoglobin abnormality may be the result of a fresh mutation because the abnormality is not present in the parents nor in any of the seven siblings.
Corpora allata from adult female Manduca sexta biosynthesize the sesquiterpenoid juvenile hormone (JH) III and the unusual ethyl-branched homologue JH II in vitro. We maintained corpora allata in medium 199 using [methyl-3H]methionine as the source of the JH methyl ester moiety and as a mass marker. This allowed measurement of the relative contributions of 14C-labeled precursors to the biogenesis of JH II and III carbon skeletons. We showed efficient incorporation of a propionate equivalent, from isoleucine or valine catabolism, into the ethyl-branched portion of JH II, using double-label liquid scintillation counting of isolated JHs and gas chromatography/mass spectrometry with selected ion monitoring of JH deuteromethoxyhydrin derivatives. Methionine was a poor source of propionate for JH II biosynthesis, while glucose, succinate, threonine, and β-alanine did not contribute propionate at all. Leucine, isoleucine, and glucose incorporated into JH III and the acetate-derived portion of JH II.
A valine tRNA was purified from Bacillus subtilis W168 by a combined use of several column chromatographic systems. The nucleotide sequence was determined to be pG-G-A-G-G-A-U-U-A-G-C-U-C-A-G-C-D-G-G-G-A-G-A-G-C-A-U-C--G-C-C-U-mo5U-A-C-m6-A -A-G-C-A-G-A-G-G-m7G-U-C-G-G-C-G-G-T-psi-C-G-A-G-C-C-C-G-U-C-A-U-C-C-U-C-C-A-C- C-AOH with the main use of of non-labeled tRNA and with the subsidiary use of [32P]-post-labeled sample. This tRNA contains 5-methoxy-uridine (mo5U) at the wobble position of the anticodon. A binding experiment of valyl-tRNA to ribosome revealed that mo5U is recognized by A and G, and fairly well by U.
Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc α-subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates). Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol. Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography. V350F produced both nitrohydroquinone at a rate of 0.75 ± 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 ± 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 ± 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 ± 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 ± 0.02 nmol/min/mg of protein...
Data are presented suggesting that intact Neurospora crassa mitochondria contain an enzyme complex incorporating five enzymes necessary for the biosynthesis of isoleucine and valine. The functional integrity and stability of the complex has been shown to be dependent on the metabolic state of the mitochondria. The complex has been solubilized by digitonin and has an approximate molecular weight of 400,000.
A microbiological method for the assay of valine, leucine, and methionine in serum is described and its accuracy and reproducibility are assessed. Under the conditions specified the L-isomers only of the three amino acids are measured. The serum levels of the three amino acids in 60 normal subjects are presented.
Minihelices mimicking the amino acid acceptor and anticodon branches of yeast tRNA(Val) have been synthesized by in vitro transcription of synthetic templates. It is shown that a minihelix corresponding to the amino acid acceptor branch and containing solely a valine-specific identity nucleotide can be aminoacylated by yeast valyl-tRNA synthetase. Its charging ability is lost after mutating this nucleotide. This ability is stimulated somewhat by the addition of a second hairpin helix that mimicks the anticodon arm, which suggests that information originating from the anticodon stem-loop can be transmitted to the active site of the enzyme by the core of the protein.
The skeleton of a child with osteogenesis imperfecta type III, resulting from the substitution of glycine 586 by valine in the triple helical domain of the alpha 2 (I) chain of type I collagen, was severely porotic but contained lamellar bone and Haversian systems. From early childhood, structural failure of the bone resulted in the disruption of growth plates, progressive bone deformities, and severe growth retardation.
A cell-free extract of Cephalosporium acremonium (Takeda N-2) was obtained that synthesized the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and also the dipeptide delta-(L-alpha-aminoadipyl)-L-cysteine from the corresponding L-amino acids.
G.l.c.--mass-spectral analysis of headspace above cultures of Aeromonas and Moraxella spp. indicates the presence of isobutyronitrile, isobutyraldoxime O-methyl ether, methacrylonitrile and possibly methacrylaldoxime O-methyl ether. Accumulation of these catabolites is maximal under low oxygen concentrations and is enhanced by enrichment of the medium with valine. Isobutyraldoxime O-methyl ether is established as the compound observed but not identified in previous studies with other bacterial species involved in spoilage of meat and chicken.
Cell-free extracts of antibiotic-negative mutants of Cephalosporium acremonium converted delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-tripeptide) into an antibiotic that was destroyed by penicillinase. The enzymic activity of the extracts was destroyed by boiling, but was not inhibited by cycloheximide. LLL-Tripeptide was totally inactive as substrate. The product resembled isopenicillin N, but not penicillin N, in its antibacterial spectrum. We propose that isopenicillin N is the first product of cyclization of LLD-tripeptide.
1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.
The properties of 22 isoleucine-valine auxotrophs induced in Escherichia coli K-12 by the transposable element, Tn5, were characterized on the basis of growth requirements, cross-feeding behavior, and enzyme activity. Mutants defective in ilvA, ilvC, ilvD and ilvE were found. Mutations in ilvE were not completely polar on ilvD and ilvA enzyme activities (that is, ilvE mutants possessed a low constitutive level of expression of the enzymes coded by ilvD and ilvA), while mutations in ilvD were completely polar on ilvA enzyme activity. The data suggest that there is an internal promoter between the sites of Tn5 insertion in ilvE and ilvD.
When the central valine residues 6, 7, and 8 of gramicidin A (gA) are shifted by one position, the resulting [Val(5), D-Ala(8)]gA forms right-handed channels with a single-channel conductance and average duration somewhat less than gA channels. The reduction in channel duration has been attributed to steric conflict between the side chains of Val(1) and Val(5) in opposing monomers (Koeppe, R. E. II, D. V. Greathouse, A. Jude, G. Saberwal, L. L. Providence, and O. S. Andersen. 1994. J. Biol. Chem. 269:12567-12576). To investigate the orientations and motions of valines in [Val(5), D-Ala(8)]gA, we have incorporated (2)H labels at Val 1, 5, or 7 and recorded (2)H-NMR spectra of oriented and nonoriented samples in hydrated dimyristoylphosphatidylcholine. Spectra of nonoriented samples at 4 degrees C reveal powder patterns that indicate rapid side chain "hopping" for Val(5), and an intermediate rate of hopping for Val(1) and Val(7) that is somewhat slower than in gA. Oriented samples of deuterated Val(1) and Val(7) show large changes in the methyl and C(beta)-(2)H quadrupolar splittings (Deltanu(q)) when Ala(5) in native gA is changed to Val(5). Three or more peaks for the Val(1) methyls with Deltanu(q) values that vary with the echo delay...
Monomolecular films of valine gramicidin A (VGA) were investigated in situ at the air-water interface by x-ray reflectivity and x-ray grazing incidence diffraction as well as polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). These techniques were combined to obtain information on the secondary structure and the orientation of VGA and to characterize the shoulder observed in its pi-A isotherm. The thickness of the film was obtained by x-ray reflectivity, and the secondary structure of VGA was monitored using the frequency position of the amide I band. The PM-IRRAS spectra were compared with the simulated ones to identify the conformation adopted by VGA in monolayer. At large molecular area, VGA shows a disordered secondary structure, whereas at smaller molecular areas, VGA adopts an anti-parallel double-strand intertwined beta(5.6) helical conformation with 30 degrees orientation with respect to the normal with a thickness of 25 A. The interface between bulk water and the VGA monolayer was investigated by x-ray reflectivity as well as by comparing the experimental and the simulated PM-IRRAS spectra on D(2)O and H(2)O, which suggested the presence of oriented water molecules between the bulk and the monolayer.
Human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, contains conserved motifs common to retroviral reverse transcriptases and telomerases. Within the C motif of hTERT is the Leu866-Val867-Asp868-Asp869 tetrapeptide that includes a catalytically essential aspartate dyad. Site-directed mutagenesis of Tyr183 and Met184 residues in HIV-1 RT, residues analogous to Leu866 and Val867, revealed that they are key determinants of nucleotide binding, processivity and fidelity. In this study, we show that substitutions at Val867 lead to significant changes in overall enzyme activity and telomere repeat extension rate, but have little effect on polymerase processivity. All Val867 substitutions examined (Ala, Met, Thr) led to reduced repeat extension rates, ranging from ∼20 to 50% of the wild-type rate. Reconstitution of V867M hTERT and telomerase RNAs (TRs) with mutated template sequences revealed the effect on extension rate was associated with a template copying defect specific to template A residues. Furthermore, the Val867 hTERT mutants also displayed increased nucleotide incorporation fidelity, implicating Val867 as a determinant of telomerase fidelity. These findings suggest that by evolving to have a valine at position 867...
1. The conductivities of aqueous solutions of glycine, d,l-valine, and l-asparagine have been determined, and comparisons have been made with similar data reported in the literature. 2. On the basis of certain theoretical considerations, calculations of the expected conductivities of aqueous solutions of glycine, asparagine, aspartic acid, and glutamic acid have been made and these data have been compared with similar data obtained experimentally. 3. The dissociation constants of the carboxyl groups of aspartic acid and glutamic acid have been calculated from conductivity data. 4. It is shown that alanine has no effect on the ionic atmosphere of solutions of potassium chloride.