RESUMEN
Endpolygalacturonase I from Stereum purpureum has been identified as a causative substance for the silver-leaf disease in apples. It possesses a unique pro-sequence in the C-terminal region that lacks endpolygalacturonases from any other origin. In this study, we analyzed and compared enzymatic characteristics between pro-form (pro-endoPG I) and mature form processed by V8 protease (endoPG I) and described the suppression activity of the pro-sequence. Of note, the optimal pH for pro-endoPG I activity shifted to pH 4.0 from pH 4.5-5.0 of endoPG I. The kinetic parameters indicated that the activity inhibition resulted from a pH-independent decrease of substrate affinity and pH-dependent deterioration of velocity by the pro-sequence. Analysis of site-directed mutations within pro-endoPG I showed that its α-helical structure includes two glutamates (E364 and E366) and alanine (A365), and its orientation by prolines (especially P348) in the pro-sequence played a significant role in its suppression activity. As for mutations in the mature domain, a marked reduction of suppression was observed for enzymes with mutations in H150, R220 and K253, indicating that the pro-sequence interacts with the active cleft by a few ionic bonds.
Asunto(s)
Agaricales/enzimología , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Poligalacturonasa/química , Poligalacturonasa/metabolismo , Agaricales/genética , Secuencia de Aminoácidos , Concentración de Iones de Hidrógeno , Cinética , Malus/microbiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pectinas/metabolismo , Poligalacturonasa/genética , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/metabolismoRESUMEN
The higher plant ADP-glucose (ADPG) pyrophosphorylase (AGPase), composed of two small subunits and two large subunits (LSs), produces ADPG, the sole substrate for starch biosynthesis from α-D-glucose 1-phosphate and ATP. This enzyme controls a key step in starch synthesis as its catalytic activity is activated by 3-phosphoglycerate (3-PGA) and inhibited by orthophosphate (Pi). Previously, two mutations in the LS of potato AGPase (PLS), PLS-E38K and PLS-G101N, were found to increase sensitivity to 3-PGA activation and tolerance to Pi inhibition. In the present study, the double mutated enzyme (PLS-E38K/G101N) was evaluated. In a complementation assay of ADPG synthesis in an Escherichia coli mutant defective in the synthesis of ADPG, expression of PLS-E38K/G101N mediated higher glycogen production than wild-type potato AGPase (PLS-WT) and the single mutant enzymes, PLS-E38K and PLS-G101N, individually. Purified PLS-E38K/G101N showed higher sensitivity to 3-PGA activation and tolerance to Pi inhibition than PLS-E38K or PLS-G101N. Moreover, the enzyme activities of PLS-E38K, PLS-G101N, and PLS-E38K/G101N were more readily stimulated by other major phosphate-ester metabolites, such as fructose 6-phosphate, fructose 2,6-bisphosphate, and ribose 5-phosphate, than was that of PLS-WT. Hence, although the specific enzyme activities of the LS mutants toward 3-PGA were impaired to some extent by the mutations, our results suggest that their enhanced allosteric regulatory properties and the broadened effector selectivity gained by the same mutations not only offset the lowered enzyme catalytic turnover rates but also increase the net performance of potato AGPase in vivo in view of increased glycogen production in bacterial cells.
Asunto(s)
Glucosa-1-Fosfato Adenililtransferasa/química , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Tubérculos de la Planta/enzimología , Solanum tuberosum/enzimología , Regulación Alostérica/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucosa-1-Fosfato Adenililtransferasa/genética , Ácidos Glicéricos/farmacología , Cinética , Modelos Moleculares , Proteínas Mutantes/genética , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
Nucleoside diphosphate kinase (NDPK) is a ubiquitous enzyme that catalyzes the transfer of the γ-phosphoryl group from a nucleoside triphosphate to a nucleoside diphosphate. In this study, we examined the subcellular localization, tissue-specific gene expression, and enzymatic characteristics of three rice NDPK isozymes (OsNDPK1-OsNDPK3). Sequence comparison of the three OsNDPKs suggested differential subcellular localization. Transient expression of green fluorescence protein-fused proteins in onion cells indicated that OsNDPK2 and OsNDPK3 are localized to plastid and mitochondria respectively, while OsNDPK1 is localized to the cytosol. Expression analysis indicated that all the OsNDPKs are expressed in the leaf, leaf sheath, and immature seeds, except for OsNDPK1, in the leaf sheath. Recombinant OsNDPK2 and OsNDPK3 showed lower optimum pH and higher stability under acidic pH than OsNDPK1. In ATP formation, all the OsNDPKs displayed lower K(m) values for the second substrate, ADP, than for the first substrate, NTP, and showed lowest and highest K(m) values for GTP and CTP respectively.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Isoenzimas/metabolismo , Nucleósido-Difosfato Quinasa/metabolismo , Cebollas/enzimología , Oryza/enzimología , Proteínas Recombinantes de Fusión/metabolismo , Adenosina Difosfato/metabolismo , Clonación Molecular , Citosol/enzimología , Estabilidad de Enzimas , Escherichia coli , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Concentración de Iones de Hidrógeno , Isoenzimas/química , Isoenzimas/genética , Cinética , Microscopía Fluorescente , Mitocondrias/enzimología , Nucleósido-Difosfato Quinasa/química , Nucleósido-Difosfato Quinasa/genética , Cebollas/citología , Cebollas/genética , Oryza/genética , Hojas de la Planta/enzimología , Plásmidos , Plastidios/enzimología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Semillas/enzimología , Especificidad por Sustrato , Transformación BacterianaRESUMEN
Tuberonic acid (TA) and its glucoside (TAG) have been isolated from potato (Solanum tuberosum L.) leaflets and shown to exhibit tuber-inducing properties. These compounds were reported to be biosynthesized from jasmonic acid (JA) by hydroxylation and subsequent glycosylation, and to be contained in various plant species. Here we describe the in vivo hydrolytic activity of TAG in rice. In this study, the TA resulting from TAG was not converted into JA. Tuberonic acid glucoside (TAG)-hydrolyzing beta-glucosidase, designated OsTAGG1, was purified from rice by six purification steps with an approximately 4300-fold purification. The purified enzyme migrated as a single band on native PAGE, but as two bands with molecular masses of 42 and 26 kDa on SDS-PAGE. Results from N-terminal sequencing and peptide mass fingerprinting of both polypeptides suggested that both bands were derived from a single polypeptide, which is a member of the glycosyl hydrolase family 1. In the native enzyme, the K(m) and V(max) values of TAG were 31.7 microM and 0.25 microkatal/mg protein, OsTAGG1 preferentially hydrolyzed TAG and methyl TAG. Here we report that OsTAGG1 is a specific beta-glucosidase hydrolyzing TAG, which releases the physiologically active TA.
Asunto(s)
Acetatos/aislamiento & purificación , Ciclopentanos/aislamiento & purificación , Glucósidos/aislamiento & purificación , Oryza/enzimología , Solanum tuberosum/química , beta-Glucosidasa/metabolismo , Acetatos/química , Acetatos/metabolismo , Ciclopentanos/química , Ciclopentanos/metabolismo , Electroforesis en Gel de Poliacrilamida , Glucósidos/genética , Glicosilación , Hidrólisis , Estructura Molecular , Oryza/metabolismo , Oxilipinas , Mapeo Peptídico , Hojas de la Planta/química , Tubérculos de la Planta/efectos de los fármacos , Plantas/química , beta-Glucosidasa/química , beta-Glucosidasa/aislamiento & purificaciónRESUMEN
Tuberonic acid (12-hydroxy epi-jasmonic acid, TA) and its glucoside (TAG) were isolated from potato leaflets (Solanumtuberosum L.) and shown to have tuber-inducing properties. The metabolism of jasmonic acid (JA) to TAG in plant leaflets, and translocation of the resulting TAG to the distal parts, was demonstrated in a previous study. It is thought that TAG generated from JA transmits a signal from the damaged parts to the undamaged parts by this mechanism. In this report, the metabolism of TA in higher plants was demonstrated using [12-(3)H]TA, and a glucosyltransferase active toward TA was purified from the rice cell cultures. The purified protein was shown to be a putative salicylic acid (SA) glucosyltransferase (OsSGT) by MALDI-TOF-MS analysis. Recombinant OsSGT obtained by overexpression in Escherichia coli was active not only toward TA but also toward SA. The OsSGT characterized in this research was not specific, but this is the first report of a glucosyltransferase active toward TA. mRNA expressional analysis of OsSGT and quantification of TA, TAG, SA and SAG after mechanical wounding indicated that OsSGT is involved in the wounding response. These results demonstrated a crucial role for TAG not only in potato tuber formation, but also in the stress response in plants and that the SA glucosyltransferase can work for TA glucosylation.
Asunto(s)
Acetatos/metabolismo , Ciclopentanos/metabolismo , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Enfermedades de las Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Acetatos/química , Línea Celular , Clonación Molecular , Ciclopentanos/química , ADN Complementario , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Glucósidos/química , Glucósidos/metabolismo , Estructura Molecular , Oryza/enzimología , Oryza/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salicilatos/metabolismo , Solanum tuberosum/enzimología , Solanum tuberosum/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The ATP binding region in the catalytically inactive large subunit (LS) of the potato tuber ADP-glucose pyrophosphorylase was identified and investigated. Mutations at the ATP binding significantly affected not only the apparent affinities for ATP and Glc-1-P, and catalytic rate but also in many instances, sensitivity to 3-phosphoglycerate. The catalytic rates of the LS mutant enzymes correlated most strongly with changes in the affinity toward ATP, a relationship substantiated by photoaffinity labeling studies with azido-ATP analog. These results indicate that the LS, although catalytically defective, interacts cooperatively with the catalytic small subunit in binding substrates and effectors and, in turn, influencing net catalysis.