Allosteric inhibition by phosphoenolpyruvate of glucose-6-phosphate dehydrogenase from bacteria and its taxonomic importance

Purified glucose-6-phosphate dehydrogenase from Zymomonas mobilis was examined with respect to inhibition by phosphoenolpyruvate, ADP and ATP. Its molecular weight was 260,000 and the kinetics of substrate conversion indicated a random bi bi mechanism. This enzyme and the dehydrogenases from Z. anaerobia, Azotobacter chroococcum, A. vinelandii, and “Corynebacterium” autotrophicum strain 19/-/x were found to be allosterically inhibited by phosphoenolpyruvate, while those from several coryneform bacteria and from Escherichia coli or Pseudomonas fluorescens were not.     

Protein expression changes during cotton fiber elongation in response to low temperature stress

Low temperature stress is one of the major abiotic stresses limiting the formation of cotton (Gossypium hirsutum L.) fiber qualities, especially fiber length. To investigate the molecular adaptation mechanisms of cotton fiber elongation to low temperature stress, two cotton cultivars, Kemian 1 (low temperature-tolerant) and Sumian 15 (low temperature-sensitive), were planted in the field at two sowing dates (25 April and 10 June). The two sowing dates resulted in different growing conditions and the main environmental difference between them was temperature, particularly the mean daily minimum temperature (MDTmin). When the sowing date was delayed, the MDTmin decreased from 26.9 °C (25 April) to 20.6 °C (10 June). Low temperature stress (MDTmin of 20.6 °C) shortened the fiber length significantly in two cultivars, but the decreased extent was larger in Sumian 15 than that in Kemian 1. Proteomic analysis of three developmental stages (10, 15 and 20 days post-anthesis [DPA]) showed that 37 spots changed significantly (p < 0.05) in abundance under low temperature stress and they were identified using mass spectrometry. These proteins were involved in malate metabolism, soluble sugar metabolism, cell wall loosening, cellulose synthesis, cytoskeleton, cellular response, and redox homeostasis. The results suggest that the enhancement of osmoticum maintenance, cell wall loosening, cell wall components biosynthesis, and cytoskeleton homeostasis plays important roles in the tolerance of cotton fibers to low temperature stress. Moreover, low levels of PEPCase, expansin, and ethylene signaling proteins may potentially lead to the low temperature sensitivity of Sumian 15 at the proteomic level.     

Kinetics of the thermal inactivation and aggregate formation of rabbit muscle pyruvate kinase in the presence of trehalose

In a previous study we found that 30-40% dimethylsulfoxide induces the active conformation of rabbit muscle pyruvate kinase. Because dimethylsulfoxide is known to perturb structure and function of many proteins, we have explored the effect of trehalose on the kinetics of thermal inactivation and stability of pyruvate kinase; this is because trehalose, in contrast to dimethyl sulfoxide, is totally excluded from the hydration shell of proteins. The results show that 600 mM trehalose inhibits the activity of pyruvate kinase by about 20% at 25 °C, however, trehalose protects pyruvate kinase from thermal inactivation at 60 °C, increases the Tm_app of unfolding by 7.2 °C, induces a more compact state, and stabilizes its tetrameric structure. The inactivation process is irreversible due to the formation of protein aggregates. Trehalose diminishes the rate of formation of intermediates with propensity to aggregate, but does not affect the extent of aggregation. Remarkably, trehalose affects the aggregation process by inducing aggregates with amyloid-like characteristics.     

Domain complementation studies reveal residues critical for the activity of the mannitol permease from Escherichia coli

This paper presents domain complementation studies in the mannitol transporter, EII^mtl, from Escherichia coli. EII^mtl is responsible for the transport and concomitant phosphorylation of mannitol over the cytoplasmic membrane. By using tryptophan-less EII^mtl as a basis, each of the four phenylalanines located in the cytoplasmic loop between putative transmembrane helices II and III in the membrane-embedded C domain were replaced by tryptophan, yielding the mutants W97, W114, W126, and W133. Except for W97, these single-tryptophan mutants exhibited a high, wild-type-like, binding affinity for mannitol. Of the four mutants, only W114 showed a high mannitol phosphorylation activity. EII^mtl is functional as a dimer and the effect of these mutations on the oligomeric activity was investigated via heterodimer formation (C/C domain complementation studies). The low phosphorylation activities of W126 and W133 could be increased 7-28 fold by forming heterodimers with either the C domain of W97 (IIC^mtlW97) or the inactive EII^mtl mutant G196D. W126 and W133, on the other hand, did not complement each other. This study points towards a role of positions 97, 126 and 133 in the oligomeric activation of EII^mtl. The involvement of specific residue positions in the oligomeric functioning of a sugar-translocating EII protein has not been presented before.     

低盐度可诱导鲈鱼胞浆型PEPCK基因表达

磷酸烯醇式丙酮酸羧激酶(PEPCK)催化草酰乙酸生成磷酸烯醇式丙酮酸,是糖异生途径的第1个限速酶.本研究用SMARTRACE技术从鲈鱼肝脏中分离克隆了PEPCK基因的全长cDNA序列.该基因全长2215bp,包含1个123bp的5′非翻译区和217bp的3′非翻译区,开放阅读框为1875bp,编码1个由624个氨基酸组成的蛋白质,该蛋白理论分子量为69.1kD,等电点为5.87.氨基酸序列分析表明,与其它动物的胞浆型PEPCK相似性很高,与黑鲷为94.2%,与大西洋鲑为86.4%,与人为75.9%,而与该鱼线粒体型PEPCK氨基酸同源性只有70.6%.系统发育分析显示,该蛋白首先与其它动物的cPEPCK聚成一支,然后再与鱼类的mPEPCK成簇,认为该PEPCK属于胞浆型.同时用RT-PCR分析了PEPCK基因在10个组织中的表达,结果表明只有在肝脏、消化道和肾脏有较高的表达.将鲈鱼从盐度为25的海水转入盐度为12的海水48h后,肝脏和肾脏的PEPCK基因表达有增加.实验结果表明,本实验克隆的为鲈鱼胞浆型PEPCK,低盐度可诱导其表达.     

CO2 Exchange and Chlorophyll Fluorescence of Phospho-enolpyruvate Carboxylase Transgenic Rice Pollen Lines

To elucidate the photosynthetic physiological characteristics and the physiological inherited traits of rice （Oryza sativa L.） hybrids and their parents, physiological indices of photosynthetic CO2 exchange and chlorophyll fluorescence parameters were measured in leaves of the maize phosphoenolpyruvate carboxylase （PEPC） transgenic rice as the male parent, sp. japonica rice cv. 9516 as the female parent, and the stable JAAS45 pollen line. The results revealed that the PEPC gene could be stably inherited and trans- ferred from the male parent to the JAAS45 pollen line. Moreover, the JAAS45 pollen line exhibited high levels of PEPC activity, manifesting higher saturated photosynthetic rates, photosynthetic apparent quantum yield （AQY）, photochemical efficiency of photosystem II and photochemical and non-photochemical quenching, which indicated that the JAAS45 pollen line has a high tolerance to photo-inhibition/photooxidation under strong light and high temperature. Furthermore, JAAS45 was confirmed to still be a C3 plant by δ^13C carbon isotope determination and was demonstrated to have a limited photosynthetic C4 microcycle by feeding with exogenous C4 primary products, such as oxaloacetate or malate, or phosphoenolpyruvate. The present study explains the physiological inherited properties of PEPC transgenic rice and provides an expectation for the integration of traditional breeding and biological technology.     

Photosynthetic and carbohydrate status of easy-and difficult-to-acclimatize sea oats (Uniola paniculata L.) genotypes during In vitro culture and Ex vitro acclimatization

Summary The photosynthetic and carbohydrate status of an easy-to-acclimatize (EK 16-3) and a difficult-to-acclimatize (EK 11-1) genotype of Uniola paniculata L. (sea oats), a native dune species of the southeastern US, were evaluated during in vitro culture and ex vitro acclimatization. Net photosynthetic rate was eight times greater for EK 16-3 than EK 11-1 plantlets after ex vitro transfer. In vitro-produced leaves were morphologically similar to ex vitro-produced leaves and exhibited similar photosynthetic competence. EK 11-1 plantlets exhibited greater transpiration rates at the time of ex vitro transfer than EK 16-3 plantlets. However, the small magnitude of this difference, although significant, indicated that control of water loss was probably not the main cause for poor acclimatization of EK 11-1 plantlets. Carbohydrate analysis in vitro revealed that EK 16-3 plantlets utilized leaf starch reserves more rapidly than EK 11-1 plantlets. Starch utilization correlated with the development of leaves with expanded leaf blades during in vitro rooting in EK 16-3 plantlets. After ex vitro transfer, both genotypes exhibited significant decreases of starch and soluble sugar content in shoots and roots. However, the higher photosynthetic ability of shoots in EK 16-3 resulted in greater accumulation of shoot soluble sugars than EK 11-1 after 2-wk ex vitro culture. After 6-wk in vitro rooting, there were significantly higher chlorophyll and soluble protein contents, ribulose 1,5-bisphosphate carboxylase (rubisco) and phosphoenolpyruvate carboxylase activities in EK 16-3 than EK 11-1 shoots. These differences also correlated with the development of anatomical and morphological leaf features in EK 16-3 similar to those of greenhouse-produced leaves.     

Characterization of two functional phosphoenolpyruvate/phosphate translocator (PPT) genes in Arabidopsis--AtPPT1 may be involved in the provision of signals for correct mesophyll development

The Arabidopsis thaliana chlorophyll a/b-binding protein underexpressed 1 (cue1) mutant shows a reticulate leaf phenotype and is defective in a plastidic phosphoenolpyruvate (PEP)/phosphate translocator (AtPPT1). A functional AtPPT1 providing plastids with PEP for the shikimate pathway is therefore essential for correct leaf development. The Arabidopsis genome contains a second PPT gene, AtPPT2. Both transporters share similar substrate specificities and are therefore able to transport PEP into plastids. The cue1 phenotype could partially be complemented by ectopic expression of AtPPT2 but obviously not by the endogeneous AtPPT2. Both genes are differentially expressed in most tissues: AtPPT1 is mainly expressed in the vasculature of leaves and roots, especially in xylem parenchyma cells, but not in leaf mesophyll cells, whereas AtPPT2 is expressed ubiquitously in leaves, but not in roots. The expression profiles are corroborated by tissue-specific transport data. As AtPPT1 expression is absent in mesophyll cells that are severely affected in the cue1 mutant, we propose that the vasculature-located AtPPT1 is involved in the generation of phenylpropanoid metabolism-derived signal molecules that trigger development in interveinal leaf regions. This signal probably originates from the root vasculature where only AtPPT1, but not AtPPT2, is present.     

Enzyme I: the gateway to the bacterial phosphoenolpyruvate:sugar phosphotransferase system.

Regulatory aspects of the bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) are reviewed. The structure and conformational stability of the first protein (enzyme I) of the PTS, as well as the requirement for enzyme I to dimerize for autophosphorylation by PEP in the presence of MgCl2 are discussed.