Ramakrishnan, T. (Yale University, New Haven, Conn.), and Edward A. Adelberg. Regulatory mechanisms in the biosynthesis of isoleucine and valine. III. Map order of the structural genes and operator genes. J. Bacteriol. 89:661–664. 1965.—A new method has been employed to determine the map order of the structural genes and operator genes governing the enzymes of the isoleucine-valine biosynthetic pathway. This method relies on the observation that phage transduction of markers carried on an F-genote leads to the establishment in the recipient of F-genotes of various lengths. Using this method, we have established that the order of loci is the following: F/ilvE ilvD ilvA oprA/ilvC/ilvB oprB. The operator locus, oprA, regulates the activity of structural genes ilvE (transaminase B), ilvD (dehydrase), and ilvA (threonine deaminase). The operator locus, oprB, regulates the activity of ilvB (condensing enzyme). An operator for ilvC (reductoisomerase) can be inferred to exist, but has not yet been detected genetically. The loci ilvB and oprB have been shown to be at the extreme right end of the sequence, but their positions relative to each other remain to be established.
The derepression of the isoleucine and valine biosynthetic enzymes in Escherichia coli and Salmonella typhimurium was examined under conditions of restriction of isoleucine, valine, or leucine (the three amino acids needed for multivalent repression of these enzymes). A procedure was used that allowed the measurement of enzyme-forming potential that accumulated during the starvation period, but could not be expressed unless the missing amino acid was supplied. The threonine deaminase (the product of the ilvA gene)-forming potential that accumulated under such conditions was found to be unstable and decayed with a half-life of about 2.5 min (at 37 C). Evidence was obtained that indicates the threonine deaminase-forming potential that accumulates under conditions of isoleucine starvation is in the form of initiated (rifampin-resistant), but uncompleted (actinomycin D-sensitive), messenger ribonucleic acid chains. Furthermore, it appears that a large portion of the threonine deaminase- and dehydrase (the product of the ilvD gene)-forming potential, under such conditions, is in the form of initiated polypeptide chains. Based on these results and results obtained with SuA− strains, a model is presented that explains how the second gene (D) in the ilvADE operon can be partially transcribed and translated under conditions in which there are no completed messenger ribonucleic acids for the gene (A) transcribed before it.
A hisT mutant of Salmonella typhimurium was found to have altered regulation of the isoleucine-valine and leucine enzymes. These enzymes in the hisT strain were derepressed two- to eightfold over those of the parent wild-type strain when grown in minimal medium or under repressing conditions. The amount of tRNALeu and the cellular concentration of charged tRNALeu was about the same in the hisT strain and in the wild type. However, leucyl-tRNA from the mutant was chromatographically different from that of wild type, confirming previous reports that hisT strains have altered tRNALeu. These results suggest strongly that tRNALeu is involved in repression of the isoleucine-valine and leucine enzymes in S. typhimurium.
A clinical isolate of Neisseria gonorrhoeae with an unusual growth requirement for isoleucine and valine lacked the activity of acetohydroxy acid synthetase, one of the enzymes required for the biosynthesis of these amino acids. A spontaneous mutant which no longer required isoleucine and valine had acquired this enzymatic activity.
Mutations in two chromosomal genes of Escherichia coli, cpxA and cpxB, produced a temperature-sensitive growth defect that was remedied specifically by the addition of isoleucine and valine to the minimal medium. This auxotrophy was manifested only when the medium contained exogenous leucine, suggesting that mutant cells fail to elaborate active acetohydroxy acid synthase, isozyme I. In the presence of leucine, this enzyme was required to catalyze the first reaction common to the biosynthesis of isoleucine and valine. Measurements of enzyme activity in crude extracts showed that mutant cells were seven- to eightfold deficient in active isozyme I when the cells were grown in the presence of leucine. When grown in the absence of leucine, mutant cells contained more acetohydroxy acid synthase activity. We attribute this activity to isozyme III, the product of the ilvHI genes, which are derepressed in the absence of exogenous leucine. The cpxA and cpxB mutations appear to affect the production of active isozyme I, rather than its activity, since (i) neither the cpxA nor the cpxB gene mapped near the structural gene for isozyme I (ilvB), (ii) the growth of mutant cells shifted from the permissive (34 degrees C) to the nonpermissive (41 degrees C) temperature did not immediately cease...
A mutant of Corynebacterium glutamicum was found to accumulate high concentrations of a material which crystallized upon cooling of the broth. The compound was identified as tetramethylpyrazine. The mutant was found to require isoleucine, valine, leucine, and pantothenate for growth. All four requirements probably result from the loss of a single enzyme of the isoleucine-valine pathway. Since similar mutants of Neurospora crassa accumulate acetoin, the present mutant probably forms tetramethylpyrazine from acetoin. Accumulation of tetramethylpyrazine was dependent upon addition of thiamine. This observation is consistent with the known activity of diphosphothiamine as a cofactor for the formation of acetolactate (a precursor of acetoin) from pyruvate.
Barat, M. (Centre National de la Recherche Scientifique, Gif-sur-Yvette, Seine et Oise, France), C. Anagnostopoulos, and A.-M. Schneider. Linkage relationships of genes controlling isoleucine, valine, and leucine biosynthesis in Bacillus subtilis. J. Bacteriol.90:357–369. 1965.—In Bacillus subtilis, the genetic loci controlling isoleucine and valine biosynthesis are not all clustered. Some of them were located on two distinct transforming deoxyribonucleic acid “molecules.” One of these molecules (the “ileilva2–4-met segment”) carries the threonine deaminase and the dihydroxy acid dehydrase loci linked to methionine markers. The other (the “ilva1–3-leu segment”) bears the reductoisomerase locus and one or more loci involved in leucine synthesis. A phenylalanine marker was also shown to be weakly linked to this latter group. In transduction mediated by phage PBS-1, these groups are transferred jointly with other gene clusters. The phage appears to convey chromosome fragments considerably longer than the transforming “molecules.” The genetic maps of both the above segments were extended by transduction. Some groups previously studied by transformation can be placed in the following linear order: the ile-ilva2–4-met segment...
l-[3,3′-3H]cystine was incorporated into penicillin with retention of one tritium. This result can be explained by β-lactam formation through ring closure between C3 of cysteine and NH of valine. No radioactivity of dl-[2,3-3H]valine was incorporated into penicillin. The loss of isotope at C2 occurs during the inversion of configuration. The loss of label at C3 is discussed in terms of possible intermediates for the formation of the thiazolidine ring of penicillin.
Cultures of Penicillium chrysogenum, growth with [35S]sulfate or labeled amino acids, were examined by ion-exchange chromatography for possible peptidic precursors of penicillin. A sulfur-containing compound, present in both the mycelial extracts and the culture filtrates, was eluted at the location of the synthetic lld-tripeptide δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine. Since this compound was also labeled when the cultures were incubated with dl-[6-14C]α-aminoadipic acid, l-[3,3′-3H]cystine, or dl-[1-14C]valine, its identity with the synthetic lld-tripeptide can be accepted. No δ-(l-α-aminoadipyl)-l-cysteine or lll-tripeptide were detected. The implications of these findings for tripeptide and penicillin biosynthesis are discussed.
[Alpha-13C]- and [alpha,beta-13C]valine were administered sequentially to a patient with methylmalonicacidemia to clarify the metabolic pathway of valine from methylmalonic acid semialdehyde to methylmalonyl-CoA. Methylmalonic acid was isolated from multiple urine samples, purified, and analyzed by 13C nuclear magnetic resonance spectroscopy. Contrary to the widely accepted view, the results show unequivocally that methylmalonic acid semialdehyde is decarboxylated to propionate before conversion to methylmalonyl CoA.
The features of three babies with perinatal lethal osteogenesis imperfecta (OI II) resulting from substitutions of glycine by valine in the triple helical domain of the alpha 1(I) chain of type I collagen were studied. The babies were heterozygous for this substitution at residue 1006 in case 1 (OI35), 973 in case 2 (OI59), and 256 in case 3 (OI7B). OI35 had the most severe clinical form, OI IIC, with premature rupture of membranes, severe antepartum haemorrhage, stillbirth, severe short limbed dwarfism, and extreme osteoporosis. OI59 was a better formed baby but was also born prematurely as a result of premature rupture of membranes and severe antepartum haemorrhage. She had the radiographic features of OI IIA. OI7B was born at term and also had the radiographic features of OI IIA. Pathological examination of the skeletons of OI35 and OI59 showed grossly deficient intramembranous and endochondral ossification. Trabecular bone was sparse in the long bones and vertebrae. The trabeculae contained a cartilage core and an overlying layer of woven bone or osteoid. The diaphyses lacked cortical bone. The periosteal fibroblasts of OI35 contained grossly distended rough endoplasmic reticulum consistent with the 53% reduction in collagen secretion by cultured dermal fibroblasts. The aorta...
A kinetic analysis was made of l-valine uptake in protoplast-derived cells (mesophyll protoplasts cultured for 6 days) and in suspension-cultured cells of tobacco (Nicotiana tabacum L., cv Xanthi). Cells from wild-type and Valr-2 mutant plants were compared. A low-Km component was found in protoplast-derived cells (Km = 45 ± 5 micromolar) as well as in suspension-cultured cells (Km = 84 ± 21 micromolar). In the mutant cells the Vmax of this component was 12- to 14-fold less than in wild-type cells. A second component (Km = 2.4 ± 0.7 millimolar) was found in suspension-cultured cells but not in protoplast-derived cells; its Vmax was the same in wild-type and mutant cells. A third component was apparently unsaturable (linear component). It was present in protoplast-derived cells but not in suspension-cultured cells, and had the same magnitude in wild-type and mutant cells. The results are discussed with reference to the uptake of l-valine in leaf tissue, in which the three kinetic components have been found simultaneously. The reduced Vmax of the low-Km component in the Valr-2 mutant, and the differential expression of the other two components in suspension-cultured cells and protoplast-derived cells indicate that the kinetically distinguishable components represent physically distinct transport systems.
The sulfonylurea herbicide chlorsulfuron blocks the biosynthesis of the amino acids valine and isoleucine in plants. Addition of these two amino acids to excised pea root (Pisum sativum L. var Alaska) cultures incubated in the presence of chlorsulfuron completely alleviates herbicide-induced growth inhibition. The site of action of chlorsulfuron is the enzyme acetolactate synthase which catalyzes the first step in the biosynthesis of valine and isoleucine. This enzyme is extremely sensitive to inhibition by chlorsulfuron having I50 values ranging from 18 to 36 nanomolar. In addition, acetolactate synthase from a wide variety of tolerant and sensitive plants species is highly sensitive to inhibition by chlorsulfuron.
The enzyme activity of purified delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase from Streptomyces clavuligerus was studied biochemically. The dependence of ACV synthetase activity on reaction parameters, including substrates, cofactors, temperature and pH, were determined, resulting in a substantially increased enzyme activity. The activity is very labile to high temperature and is also unstable at acidic pH. The enzyme specificity is strict towards L-alpha-aminoadipate, but rather loose with respect to L-valine; certain modifications of L-cysteine can also be tolerated. Some unnatural tripeptides synthesized by ACV synthetase can be converted into bioactive compounds by isopenicillin N synthase. The only nutrient found to negatively affect ACV synthetase activity is phosphate, but various compounds such as thiol-blocking reagents and ATP-utilization products (AMP and pyrophosphate) are inhibitory to the enzyme.
During valine catabolism in muscle both 2-oxoisovalerate and 3-hydroxyisobutyrate can be released into the circulation. 3-Hydroxyisobutyrate is a good gluconeogenic substrate in isolated cortical tubules and hepatocytes. The maximal rate of gluconeogenesis from 3-hydroxyisobutyrate was greater than from 2-oxoisovalerate. We propose that 3-hydroxyisobutyrate is an inter-organ metabolite by which the gluconeogenic potential of valine, whose catabolism has been initiated in muscle, may be conserved.
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.
Hindquarters from starved rats were perfused with plasma concentrations of amino acids, but without other added substrates. Release of amino acids was similar to that previously reported, but, if total amino acid changes were recorded, alanine and glutamine were not formed in excess of their occurrence in muscle proteins. In protein balance (excess insulin) there was no net formation of either alanine or glutamine, even though the branched-chain amino acids and methionine were consumed. If [U-14C]valine was present, radiolabelled 3-hydroxyisobutyrate and, to a lesser extent, 2-oxo-3-methylbutyrate accumulated and radiolabel was incorporated into citrate-cycle intermediates and metabolites closely associated with the citrate cycle (glutamine and glutamate, and, to a smaller extent, lactate and alanine). If a 2-chloro-4-methylvalerate was present to stimulate the branched-chain oxo acid dehydrogenase, flux through this step was accelerated, resulting in increased accumulation of 3-hydroxyisobutyrate, decreased accumulation of 2-oxo-3-methylbutyrate, and markedly increased incorporation of radiolabel (specific and total) into all measured metabolites formed after 3-hydroxyisobutyrate. It is concluded that: amino acid catabolism by skeletal muscle is confined to degradation of the branched-chain amino acids...
1. The intracellular concentrations of sodium, potassium, ATP and creatine phosphate in chopped tissue from guinea-pig cerebral cortex were altered by a 10min period of electrical stimulation. 2. The ability of the tissue to take up l-[14C]valine and incorporate it into protein was tested at various times after the cessation of electrical stimulation. 3. The restoration of the ability of the tissue to accumulate l-[14C]valine correlated closely with the restoration of the resting intracellular concentrations of sodium and ATP.
1. Superior cervical ganglia from the guinea pig carry out an energy-dependent incorporation of l-[14C]valine into protein in vitro. 2. Stimulation of the preganglionic nerve at a physiological frequency for more than a few minutes decreases the ability of the ganglia to incorporate labelled valine into protein.
Acetohydroxyacid synthase (EC 184.108.40.206, also known as acetolactate synthase) isoenzyme II from Escherichia coli is inhibited by sulphonylurea and imidazolinone herbicides, although it is much less sensitive than the plant enzyme. This isoenzyme is also unusual in that it is not inhibited by valine. Mutating S100 (Ser(100) in one-letter amino acid notation) of the catalytic subunit to proline increases its sensitivity to sulphonylureas, but not to imidazolinones. Mutating P536 to serine, as found in the plant enzyme, had little effect on the properties of the enzyme. Mutating E14 of the regulatory subunit to glycine, either alone or in combination with the H29N (His(29)-->Asn) change, did not affect valine-sensitivity.