Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We fused four mutant omr1 alleles, encoding feedback-insensitive forms of Arabidopsis thaliana biosynthetic threonine dehydratase/deaminase (TD), to the CaMV 35S promoter and transformed these constructs into A. thaliana Columbia wild type plants. The mutant TD forms consisted of our previously isolated double mutant, omr1-1 , and three new site-directed mutants, omr1-5 , omr1-7 , and omr1-8 with single point mutations. We employed site-directed mutagenesis to assay the effects of amino acid substitutions in separate regulatory regions within the carboxy-terminal (C-term) allosteric end. TD assays and growth resistance to the isoleucine (Ile) toxic analog -O-methylthreonine (OMT) confirmed the desensitization to feedback inhibition and the viability of these mutant omr1 alleles as selectable markers, respectively. Two of the site-directed mutants, omr1-5 and omr1-7 , appeared to influence one of the two separate Ile-binding sites and had a notable 13-fold and 15-fold increase in free Ile, respectively. The omr1-8 appeared to influence the other Ile-binding site and resulted in a 2-fold increase in free Ile. The transgenic omr1-1 double mutant affecting both Ile-binding sites, however, displayed a 106-fold increase in free Ile revealing a profound synergistic interplay between these separate Ile-binding sites. While all of the four omr1 alleles conferred resistance to elevated concentrations of OMT, the progeny of omr1-1 initial transformants exhibited a bushy phenotype at the rosette stage. On the other hand, progeny of transformants omr1-5 , omr1-7 , and omr1-8 had a normal phenotype, undistinguishable from wild type. Therefore, alleles omr1-5 , omr1-7 , and omr1-8 , proved to be ideal as environmentally-friendly, dominant, selectable markers for plant transformation.
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PMID:A site-directed mutagenesis interrogation of the carboxy-terminal end of Arabidopsis thaliana threonine dehydratase/deaminase reveals a synergistic interaction between two effector-binding sites and contributes to the development of a novel selectable marker. 1560 69

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of alpha-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.
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PMID:Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate. 1666 62

Threonine deaminase (TD) catalyzes the conversion of Thr to alpha-keto butyrate in Ile biosynthesis; however, its dramatic upregulation in leaves after herbivore attack suggests a role in defense. In Nicotiana attenuata, strongly silenced TD transgenic plants were stunted, whereas mildly silenced TD transgenic plants had normal growth but were highly susceptible to Manduca sexta attack. The herbivore susceptibility was associated with the reduced levels of jasmonic acid-isoleucine (JA-Ile), trypsin proteinase inhibitors, and nicotine. Adding [(13)C(4)]Thr to wounds treated with oral secretions revealed that TD supplies Ile for JA-Ile synthesis. Applying Ile or JA-Ile to the wounds of TD-silenced plants restored herbivore resistance. Silencing JASMONATE-RESISTANT4 (JAR4), the N. attenuata homolog of the JA-Ile-conjugating enzyme JAR1, by virus-induced gene silencing confirmed that JA-Ile plays important roles in activating plant defenses. TD may also function in the insect gut as an antinutritive defense protein, decreasing the availability of Thr, because continuous supplementation of TD-silenced plants with large amounts (2 mmol) of Thr, but not Ile, increased M. sexta growth. However, the fact that the herbivore resistance of both TD- and JAR-silenced plants was completely restored by signal quantities (0.6 mumol) of JA-Ile treatment suggests that TD's defensive role can be attributed more to signaling than to antinutritive defense.
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PMID:Silencing threonine deaminase and JAR4 in Nicotiana attenuata impairs jasmonic acid-isoleucine-mediated defenses against Manduca sexta. 1708 87

Amino acids are not only fundamental protein constituents but also serve as precursors for many essential plant metabolites. Although amino acid biosynthetic pathways in plants have been identified, pathway regulation, catabolism, and downstream metabolite partitioning remain relatively uninvestigated. Conversion of Thr to Gly and acetaldehyde by Thr aldolase (EC 4.1.2.5) was only recently shown to play a role in plant amino acid metabolism. Whereas one Arabidopsis thaliana Thr aldolase (THA1) is expressed primarily in seeds and seedlings, the other (THA2) is expressed in vascular tissue throughout the plant. Metabolite profiling of tha1 mutants identified a >50-fold increase in the seed Thr content, a 50% decrease in seedling Gly content, and few other significant metabolic changes. By contrast, homozygous tha2 mutations cause a lethal albino phenotype. Rescue of tha2 mutants and tha1 tha2 double mutants by overproduction of feedback-insensitive Thr deaminase (OMR1) shows that Gly formation by THA1 and THA2 is not essential in Arabidopsis. Seed-specific expression of feedback-insensitive Thr deaminase in both tha1 and tha2 Thr aldolase mutants greatly increases seed Ile content, suggesting that these two Thr catabolic enzymes compete for a common substrate pool.
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PMID:Two Arabidopsis threonine aldolases are nonredundant and compete with threonine deaminase for a common substrate pool. 1717 52

Sebacina vermifera, a growth-promoting endophytic fungus, significantly increases Nicotiana attenuata's growth but impairs both its herbivore resistance and its accumulation of the costly, jasmonic acid (JA)-regulated defense protein, trypsin proteinase inhibitor (TPI). To determine if the fungi's growth-promoting effects can be attributed to lower TPI-related defense costs, we inoculated transformed N. attenuata plants silenced in their ability to synthesize JA, JA-isoleucine, and TPI by antisense (lipoxygenase 3 [as-lox3] and Thr deaminase [as-td]) and inverted repeat (ir-tpi) expression, and found that inoculation promoted plant growth as in untransformed wild-type plants. Moreover, herbivore-elicited increases in JA and JA-isoleucine concentrations did not differ between inoculated and uninoculated wild-type plants. However, inoculation significantly reduced the morphological effect of 1-aminocyclopropane-1-carboxylic acid on wild-type seedlings in a triple response assay, suggesting that ethylene signaling was impaired. Furthermore, S. vermifera failed to promote the growth of N. attenuata plants transformed to silence ethylene production (1-aminocyclopropane-1-carboxylic acid oxidase [ir-aco]). Inoculating wild-type plants with S. vermifera decreased the ethylene burst elicited by applying Manduca sexta oral secretions to mechanical wounds. Accordingly, oral secretion-elicited transcript levels of the ethylene synthesis genes NaACS3, NaACO1, and NaACO3 in inoculated plants were significantly lower compared to these levels in uninoculated wild-type plants. Inoculation accelerated germination in wild-type seeds; however, uninoculated wild-type seeds germinated as rapidly as inoculated seeds in the presence of the ethylene scrubber KMnO(4). In contrast, neither inoculation nor KMnO(4) exposure influenced the germination of ir-aco seeds. We conclude that S. vermifera increases plant growth by impairing ethylene production independently of JA signaling and TPI production.
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PMID:Sebacina vermifera promotes the growth and fitness of Nicotiana attenuata by inhibiting ethylene signaling. 1741 38

Plant defense against insect herbivores is mediated in part by enzymes that impair digestive processes in the insect gut. Little is known about the evolutionary origins of these enzymes, their distribution in the plant kingdom, or the mechanisms by which they act in the protease-rich environment of the animal digestive tract. One example of such an enzyme is threonine (Thr) deaminase (TD), which in tomato (Solanum lycopersicum) serves a dual role in isoleucine (Ile) biosynthesis in planta and Thr degradation in the insect midgut. Here, we report that tomato uses different TD isozymes to perform these functions. Whereas the constitutively expressed TD1 has a housekeeping role in Ile biosynthesis, expression of TD2 in leaves is activated by the jasmonate signaling pathway in response to herbivore attack. Ingestion of tomato foliage by specialist (Manduca sexta) and generalist (Trichoplusia ni) insect herbivores triggered proteolytic removal of TD2's C-terminal regulatory domain, resulting in an enzyme that degrades Thr without being inhibited through feedback by Ile. This processed form (pTD2) of TD2 accumulated to high levels in the insect midgut and feces (frass). Purified pTD2 exhibited biochemical properties that are consistent with a postingestive role in defense. Shotgun proteomic analysis of frass from tomato-reared M. sexta identified pTD2 as one of the most abundant proteins in the excrement. Among the other tomato proteins identified were several jasmonate-inducible proteins that have a known or proposed role in anti-insect defense. Subtilisin-like proteases and other pathogenesis-related proteins, as well as proteins of unknown function, were also cataloged. We conclude that proteomic analysis of frass from insect herbivores provides a robust experimental approach to identify hyperstable plant proteins that serve important roles in defense.
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PMID:Stability of plant defense proteins in the gut of insect herbivores. 1741 43

Upon wounding or infection, a serine proteinase cascade in insect hemolymph leads to prophenoloxidase (proPO) activation and melanization, a defense response against invading microbes. In the tobacco hornworm Manduca sexta, this response is initiated via hemolymph proteinase 14 (HP14), a mosaic protein that interacts with bacterial peptidoglycan or fungal beta-1,3-glucan to autoactivate. In this paper, we report the expression, purification, and functional analysis of M. sexta HP21 precursor, an HP14 substrate similar to Drosophila snake. The recombinant proHP21 is a 51.1 kDa glycoprotein with an amino-terminal clip domain, a linker region, and a carboxyl-terminal serine proteinase domain. HP14, generated by incubating proHP14 with beta-1,3-glucan and beta-1,3-glucan recognition protein-2, activated proHP21 by limited proteolysis between Leu(152) and Ile(153). Active HP21 formed an SDS-stable complex with M. sexta serpin-4, a physiological regulator of the proPO activation system. We determined the P1 site of serpin-4 to be Arg(355) and, thus, confirmed our prediction that HP21 has trypsin-like specificity. After active HP21 was added to the plasma, there was a major increase in PO activity. HP21 cleaved proPO activating proteinase-2 precursor (proPAP-2) after Lys(153) and generated an amidase activity, which activated proPO in the presence of serine proteinase homolog-1 and 2. In summary, we have discovered and reconstituted a branch of the proPO activation cascade in vitro: beta-1,3-glucan recognition--proHP14 autoactivation--proHP21 cleavage--PAP-2 generation--proPO activation--melanin formation.
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PMID:Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor. 1778 89

Systemins and their hydroxyproline-rich glycopeptide systemin (ppHS) subfamily members are known to mediate antiherbivore defenses in some solanaceous taxa but not others; functions other than in defense remain largely unexplored. Nicotiana attenuata's ppHS is known not to function in herbivore defense. NappHS transcripts are abundant in flowers, particularly in pistils, and when two N. attenuata accessions from Utah and Arizona were transformed to silence NappHS by RNAi (IRsys), seed capsule production and seed number per capsule were reduced in both accessions. These reductions in reproductive performance could not be attributed to impaired pollen or ovule viability; hand-pollination of all IRsys lines of both accessions restored seed production per capsule to levels found in wild-type plants. Rather, changes in flower morphology that decreased the efficiency of self-pollination are likely responsible: IRsys plants of both accessions have flowers with pistils that protrude beyond their anthers. Because these changes in flower morphology are reminiscent of CORONATINE-INSENSITIVE1-silenced N. attenuata plants, we measured jasmonates (JAs) and their biosynthetic transcripts in different floral developmental stages, and found levels of JA-isoleucine (Ile)/leucine and threonine deaminase transcripts, which are abundant in wild-type pistils, to be significantly reduced in IRsys buds and flowers. Threonine deaminase supplies Ile for JA-Ile biosynthesis, and we propose that ppHS mediates JA signaling during flower development and thereby changes flower morphology. These results suggest that the function of ppHS family members in N. attenuata may have diversified to modulate flower morphology and thereby outcrossing rates in response to biotic or abiotic stresses.
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PMID:Silencing the hydroxyproline-rich glycopeptide systemin precursor in two accessions of Nicotiana attenuata alters flower morphology and rates of self-pollination. 1921 1

Two proteins from the amidohydrolase superfamily of enzymes were cloned, expressed, and purified to homogeneity. The first protein, Cc0300, was from Caulobacter crescentus CB-15 (Cc0300), while the second one (Sgx9355e) was derived from an environmental DNA sequence originally isolated from the Sargasso Sea ( gi|44371129 ). The catalytic functions and the substrate profiles for the two enzymes were determined with the aid of combinatorial dipeptide libraries. Both enzymes were shown to catalyze the hydrolysis of l-Xaa-l-Xaa dipeptides in which the amino acid at the N-terminus was relatively unimportant. These enzymes were specific for hydrophobic amino acids at the C-terminus. With Cc0300, substrates terminating in isoleucine, leucine, phenylalanine, tyrosine, valine, methionine, and tryptophan were hydrolyzed. The same specificity was observed with Sgx9355e, but this protein was also able to hydrolyze peptides terminating in threonine. Both enzymes were able to hydrolyze N-acetyl and N-formyl derivatives of the hydrophobic amino acids and tripeptides. The best substrates identified for Cc0300 were l-Ala-l-Leu with k(cat) and k(cat)/K(m) values of 37 s(-1) and 1.1 x 10(5) M(-1) s(-1), respectively, and N-formyl-l-Tyr with k(cat) and k(cat)/K(m) values of 33 s(-1) and 3.9 x 10(5) M(-1) s(-1), respectively. The best substrate identified for Sgx9355e was l-Ala-l-Phe with k(cat) and k(cat)/K(m) values of 0.41 s(-1) and 5.8 x 10(3) M(-1) s(-1). The three-dimensional structure of Sgx9355e was determined to a resolution of 2.33 A with l-methionine bound in the active site. The alpha-carboxylate of the methionine is ion-paired to His-237 and also hydrogen bonded to the backbone amide groups of Val-201 and Leu-202. The alpha-amino group of the bound methionine interacts with Asp-328. The structural determinants for substrate recognition were identified and compared with other enzymes in this superfamily that hydrolyze dipeptides with different specificities.
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PMID:Functional annotation of two new carboxypeptidases from the amidohydrolase superfamily of enzymes. 1935 46

Nicotiana attenuata has the capacity to respond specifically to herbivory by its natural herbivore, Manduca sexta, through the perception of elicitors in larval oral secretions. We demonstrate that Lectin receptor kinase 1 (LecRK1) functions during M. sexta herbivory to suppress the insect-mediated inhibition of jasmonic acid (JA)-induced defense responses. Gene function analysis performed by reducing LecRK1 expression in N. attenuata by both virus-induced gene silencing and inverted repeated RNA interference (ir-lecRK1 plants) revealed that LecRK1 was essential to mount a full defense response against M. sexta folivory; larvae growing on ir-lecRK1 plants were 40 to 100% larger than those growing on wild-type plants. The insect-induced accumulation of nicotine, diterpene-glucosides, and trypsin protease inhibitors, as well as the expression of Thr deaminase, was severalfold reduced in ir-lecRK1 plants compared with the wild type. The accumulation of JA and JA-Ile was unaffected during herbivory in ir-lecRK1 plants; however, salicylic acid (SA) accumulation was increased by twofold. The expression of nahG in ir-lecRK1 plants prevented the increased accumulation of SA and restored the defense response against M. sexta herbivory. The results suggest that LecRK1 inhibits the accumulation of SA during herbivory, although other mechanisms may also be affected.
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PMID:Nicotiana attenuata LECTIN RECEPTOR KINASE1 suppresses the insect-mediated inhibition of induced defense responses during Manduca sexta herbivory. 2192 34


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