In addition to AtCPL1-4, the genome of Arabidopsis thaliana encodes a large number of putative acid phosphatases. The predicted Arabidopsis SCP1-like small phosphatases (SSP) are highly homologous to the catalytic domain of eukaryotic RNA polymerase II carboxyl terminal domain (pol II CTD) phosphatases. Among the family members, SSP4, SSP4b and SSP5 form a unique group characterized by long N-terminal extensions. These three SSPs showed similar and ubiquitous gene expression. SSP4 and SSP4b were localized exclusively in the nuclei, while SSP5 accumulated both in the nucleus and cytoplasm. In vitro observation revealed that SSP4 and SSP4b dephosphorylated the pol II CTD-PO4 at both Ser2 and Ser5 in the conserved heptad repeats; however, SSP5 dephosphorylated only Ser5 of CTD-PO4. These results indicate that Arabidopsis SSP family encodes active CTD phosphatases similarly to animal SCP1 family proteins and plant CPLs family proteins, but with distinct substrate specificities. ssp mutants did not exhibit phenotypic abnormalities under normal growth conditions. However, ssp5 single mutants and ssp4 ssp4b ssp5 triple mutants showed enhanced sensitivity to ABA and glucose during seed germination. Yet, increased ABA-inducible gene expressions were not distinguishable in triple mutants compared to wild type plants upon ABA treatment. Unlike the ssp mutations, the cpl1 mutation strongly induced RD29A expression in response to cold, ABA and NaCl treatments. Thus, the cpl1 mutant is an ideal genetic background for an inducible gene expression system, in which the detrimental effect to host plants caused by a conventional constitutive expression could be avoided. Production of flavonoid such as anthocyanins in Arabidopsis is relatively easy to monitor and is regulated by transcription factors such as PAP1. PAP1 activates the expression of multiple enzymes in the anthocyanin biosynthesis pathway; however, high level of flavonoid production could cause vegetative growth retardation. To optimize flavonoid accumulation, a three-component system was designed consisting of a cold inducible RD29A-PAP1 expression cassette, a feedforward effector RD29A-CBF3, and a mutation in host repressor CPL1. Transgenic cpl1 plants containing both homozygous PAP1 and CBF3 transgenes produced 30-fold higher level of total anthocyanins than control plants upon cold treatment. LC/MS/MS analysis showed the flavonoid profile in cold-induced transgenic plants resembled that of previously reported pap1-D plants but were enriched for kaempferol derivatives. Furthermore, PAP1 and environmental signals synergistically regulate flavonoid pathway to produce a flavonoid blend that has not been produced by PAP1 overexpression or cold treatment alone. These results delineate the usability of the three-component inducible system in plant metabolic engineering.

None

The roles of phosphorus (P) starvation on the induction of intracellular acid phosphatase (ACP; EC 3.1.3.2) activity have been studied in a marine macroalga Ulva lactuca L. In comparison to creasy and dark green appearance in P-sufficient thalli (100 mM NaH2PO4), P-starved thalli (1 mM NaH2PO4) showed less crease and light green appearance. On exposure to 1 mM NaH2PO4, the growth rate, the contents of SRP, PP and P, and tissue C:P and N:P molar ratio decreased at day 3 and the contents of SNRP, TSP and polyphosphate decreased immediately. Intracellular ACP activity increased at day 3 after exposure to 1 mM NaH2PO4 and reached 16 folds of P-sufficient thalli at day 14, while extracellular alkaline phosphatase (AP; EC 3.1.3.1) activity increased at day 2 and reached the plateau after 4 days. Activity staining both on Native PAGE and IEF gel showed the induction of 10 and 9 ACP isoenzymes, respectively. Changes in intracellular ACP and extracellular AP activities were negatively correlated with SRP, SNRP, PP and P contents. After transferred to 100 mM NaH2PO4, the growth rate of 10 day-starvated thalli recovered after 5 days, the contents of SRP¡BSNRP¡BTSP and P, and the C:P and N:P molar ratio recovered to the level of P-sufficient thalli at day 1. When recovered to 100 mM NaH2PO4, extracellular AP activity of 10 day-starvated thalli decreased at day 2 and reached the minimum after day 8, while intracellular ACP activity decreased at day 3 and reached the minimum after day 8. The analog of Pi, Phi (1 mM) inhibited the intracellular ACP and extracellular AP activities induced by P swtarvation. The results of present investigation show that ACP has a role in the enhancement of P availability in U. lactuca via the enzymatically degradation of polyphosphates and organic P when suffers P deficiency.

Thèse d'exercice : Pharmacie : Université de Nantes : 2004. / Bibliogr. f. 111-118 [63 réf.].

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "August 2003." Typescript. Vita. Includes bibliographical references (leaves 158-174). Also issued on the Internet.

Thesis (Ph. D.) in Biochemistry and Molecular Biology--University of Maine, 2001. / Includes vita. Includes bibliographical references (leaves 113-165).

Plants possess integrated signalling networks that mediate the responses to various environmental conditions. Mitogen-activated protein kinases (MAPKs) constitute a highly conserved family of enzymes in eukaryotes, and in plants MAPK-based signal transduction modules regulate a large number of physiological processes, including responses to environmental stresses and phytohormones. Regulated dephosphorylation of active MAPKs is a key component of the control of MAPK signalling cascades, and in mammals, members of the MAPK phosphatase (MKP) sub-class of dual-specificity tyrosine phosphatases have been recognized as key players for inactivating MAPKs. Five MKP homologues are found in Arabidopsis thaliana, but only limited information is available concerning their properties and biological roles. Based on initial data derived from my reverse genetics and protein interaction studies of these five potential MKPs, as well as gene function information in the literature, I chose to focus on two putative Arabidopsis MKPs, AtMKP2 and Indole-3-Butyric Acid-response 5 (IBR5). By using a combination of genetic and biochemical studies, I established that the previously uncharacterized MKP designated AtMKP2, participates in the regulation of cellular homeostasis in ozone-challenged tissue, and can influence the activation state of two MAPKs, MPK3 and MPK6. AtMKP2-suppressed plants displayed significantly prolonged MPK3 and MPK6 activation during ozone treatment, and recombinant AtMKP2 was able to dephosphorylate both phospho-MPK3 and phospho-MPK6 in vitro, providing direct evidence that AtMKP2 may target these oxidant-activated MAPKs. A mutation in IBR5, one of the five potential AtMKPs, was previously reported to confer reduced sensitivity to auxin and ABA in Arabidopsis. My protein interaction studies demonstrated that IBR5 and MPK12 are physically coupled and that the C-terminus of MPK12 is essential for its interaction with IBR5. In vitro dephosphorylation assays indicated that recombinant phosphoMPK12 is efficiently dephosphorylated by IBR5. In transgenic plants with reduced expression of the MPK12 gene, root growth is hypersensitive to exogenous auxins, consistent with the lower auxin sensitivity reported for ibr5 mutants. Taken together, my data demonstrate for the first time that both AtMKP2 and IBR5 are bona fide Arabidopsis MAPK phosphatases and that they serve as important regulators of oxidative stress and auxin signalling, respectively, in Arabidopsis.

Acid phosphatases (APases; E.C. 3.1.3.2) catalyze the hydrolysis of phosphate (Pi) from Pi monoesters and anhydrides within the acidic pH range. Induction of intracellular and secreted purple acid phosphatases (PAPs) is a widespread plant response to nutritional Pi-deficiency. The probable function of intracellular APases is to recycle Pi from expendable intracellular organophosphate pools, whereas secreted APases likely scavenge Pi from the organically bound Pi that is prevalent in most soils. Although the catalytic function and regulation of plant PAPs have been described, their physiological function in plants has not been fully established. Recent biochemical and proteomic studies indicated that AtPAP26 is the predominant intracellular (vacuolar) and a major secreted purple APase isozyme upregulated by Pi-starved (-Pi) Arabidopsis thaliana. The in planta function of AtPAP26 was assessed by molecular, biochemical, and phenotypic characterization of a homozygous Salk T-DNA insertion mutant. Loss of AtPAP26 expression resulted in the elimination of AtPAP26 transcripts and 55-kDa immunoreactive AtPAP26 polypeptides, correlated with a 9- and 5-fold decrease in extractable shoot and root APase activity, respectively, as well as a 40% reduction in secreted APase activity of –Pi seedlings. The results corroborate previous findings implying that AtPAP26 is: (i) the principal contributor to Pi starvation inducible APase activity in Arabidopsis, and (ii) controlled post-transcriptionally mainly at the level of protein accumulation. Total shoot free Pi level was about 40% lower in –Pi atpap26 mutants relative to wild-type controls, but unaffected under Pi-sufficient conditions. Moreover, shoot, root, inflorescence, and silique development of the atpap26 mutant was impaired during Pi deprivation, but unaffected under Pi-replete conditions, or during nitrogen or potassium-limited growth, or oxidative stress. The results suggest that the hydrolysis of Pi from organic-phosphate esters by AtPAP26 makes an important contribution to Pi-recycling and scavenging in –Pi Arabidopsis. / Thesis (Master, Biology) -- Queen's University, 2009-09-01 09:46:39.302

<p>We present a computational structure-based redesign of the phenylalanine adenylation domain of the non-ribosomal peptide synthetase (NRPS) enzyme gramicidin S synthetase A (GrsA-PheA) for a set of non-cognate substrates for which the wild-type enzyme has little or virtually no specificity. Experimental validation of a set of top-ranked computationally-predicted enzyme mutants shows significant improvement in the specificity for the target substrates. We further present enhancements to the methodology for computational enzyme redesign that are experimentally shown to result in significant additional improvements in the target substrate specificity. The mutant with the highest activity for a non-cognate substrate exhibits 1/6 of the wild-type enzyme/wild-type substrate activity, further confirming the feasibility of our computational approach. Our results suggest that structure-based protein design can identify active mutants different from those selected by evolution.</p><p>Knowledge about the structures of individual domains and domain interactions can further our redesign of the NRPS enzymes for new bioactive nature product. So far, little structure information has been available for the auxiliary domains such as the epimerization domains and how they interact with the NRPS modules. Solution structure studies by nuclear magnetic resonance (NMR) provide advantages for understanding the dynamics of the domains and reveal active conformations that sometimes are not represented by the crystal structures. However, the large size of the NRPS proteins present challenges for structure studies in solution. In chapter 3, we study the solution structure of the 56 kDa epimerization domain of GrsA (GrsA-PheE) by NMR. We use multidimensional backbone resonance experiments as well as specific labeling strategy to assign the backbone resonances of GrsA-PheE. Secondary structures are determined by sets of residual dipolar couplings (RDCs) measured in multiple alignment media. To determine the global fold of the protein, we obtain long-range distance restraints by measuring the paramagnetic relaxation enhancements (PREs) from 15 site-directed spin labeling samples. </p><p>In chapter 4, we investigate the redox regulation of phosphatases. The activity levels of protein tyrosine phosphatases (PTPs) in cells are highly regulated in various ways including by phosphorylation, localization and protein-protein interaction. Additionally, redox-dependent modification has emerged as a critical part in attenuating PTPs activity in response to cellular stimuli. The tandem Src homology 2 domain-containing PTPs (SHPs) belong to the family of nonreceptor PTPs. The activity level of SHPs is highly regulated by interaction of SH2 domain, phosphorylation level of C-terminal tail and by reversible oxidation. In vivo evidence has shown the reversible oxidation of catalytic cysteine inhibits SHPs activity transiently as a result, affecting the phosphorylation level of its target proteins. In this chapter, we investigate in vitro the reversible oxidation of full-length and catalytic domain of SHP-1 and SHP-2 by using kinetic measurements and mass spectrometry. We have confirmed the susceptibility of the active site cysteines of SHPs to oxidative inactivation, with rate constants for oxidation similar to other PTPs (2-10 M-1s-1). Both SHP-1 and SHP-2 can be reduced and reactivated with the reductants DTT and gluthathione, whereas only the catalytic domain of SHP-2 is subject to reactivation by thioredoxin. Unlike PTPs whose oxidation contains a catalytic cysteine disulfide bonding to a backdoor cysteine or forms a sulfenylamide bonding to nearby backbone nitrogen, we have found that in the reversibly oxidized SHPs, the catalytic cysteines is re-reduced while two conserved backdoor cysteines form a disulfide linkage. Knocking out either of the backdoor cysteine preserves the reversibility of the oxidized SHPs with a disulfide formation between the catalytic cysteine and the remaining backdoor cysteine. However, removal of both backdoor cysteines leads to irreversible oxidative inactivation, demonstrating that these two cysteines are necessary and sufficient for ensuring reversible oxidation of the SHPs. Our results extend the mechanisms by which redox regulation of PTPs is used to modulate intracellular signaling pathways.</p> / Dissertation

A continuous flow microwave heating system was set up by using one domestic microwave oven (1000W nominal output at 2450MHz). Water was run through the coil centrally located inside the oven cavity for microwave heating. Microwave absorption efficiency was evaluated by measuring inlet and outlet temperatures of coil as a function of system variables. In order to optimize the coil configuration, the influence of tube diameter (6.4, 7.9 and 9.7mm); initial temperature (10, 20 and 30 ºC); number of turns (3.5, 4.5 and 5.5); coil diameter (61.5, 88, 102, 121 and 153 mm) and pitch (16, 18, 20, 22 and 24mm) were evaluated, respectively at different flow rates (240, 270, 300, 330 and 360ml/min). In helical systems, Dean number is used as a measure of secondary flow which enhances mixing of the fluid providing uniform heating even under laminar flow conditions. Results showed that microwave absorption efficiency was a compromise between coil volume and Dean number. Therefore, a helical coil (110 mm high) with a coil diameter of 108 mm, tube diameter of 8.2 mm, 5.5 turns demonstrated the highest efficiency, fast heating rate, more uniform heating and less temperature fluctuations. The optimized coil configuration parameters were used subsequently to set up continuous-flow microwave heating system.