Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.2.2.10 (PNL)
 341 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The purposes of this study are to develop an in vivo cell system that is suitable for the immunofluorescent detection of transiently expressed proteins targeted to plant peroxisomes and to determine whether a C-terminal serine-lysine-leucine (SKL) tripeptide, a consensus-targeting signal for mammalian peroxisomes, also targets proteins to plant peroxisomes. Protoplasts from mesophyll cells and from suspension-cultured cells initially were examined for their potential as an in vivo import system. Several were found suitable, but based on a combination of criteria, suspension-cultured tobacco (Nicotiana tabacum L. cv Bright Yellow 2) cells (TBY-2) were chosen. The tobacco cell extracts had catalase activity, and two polypeptides of approximately 55 and 57 kD specifically were detected on immunoblots with anti-cottonseed catalase immunoglobulins G as the probe. Indirect immunofluorescence microscopy with these immunoglobulins G revealed a punctate labeling pattern indicative of endogenous catalase localization within putative TBY-2 peroxisomes. The cells did not have to be completely converted to protoplasts for optimal microscopy; treatment with 0.1% (w/v) pectolyase for 2 h was sufficient. Microprojectile bombardment proved superior for transient transformation of the TBY-2 cells with plasmids encoding beta-glucuronidase, or chloramphenicol acetyltransferase (CAT), or CAT with an added C-terminal tripeptide (CAT-SKL). C-terminal SKL is a consensus, type 1, peroxisome targeting signal. Double indirect immunofluorescent labeling showed that CAT-SKL co-localized with endogenous catalase. Non-punctate, diffuse localization of CAT without SKL provided direct evidence that the C-terminal SKL tripeptide was necessary and sufficient for targeting of CAT to plant peroxisomes. These data demonstrate the effectiveness of this peroxisome targeting signal for plant cells.
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PMID:Development and application of an in vivo plant peroxisome import system. 777 May 24

Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed beta-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH. On the basis of the three-dimensional structures of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677-689] and the modelled enzyme-substrate complex of PL1B [Herron, Benen, Scavetta, Visser and Jurnak (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 8762-8769], Asp154, Arg176, Arg236 and Lys239 were mutagenized. Substituting Arg236 with alanine or lysine rendered the enzyme completely inactive, and mutagenesis of Arg176 and Lys239 severely affected catalysis. The Asp154-->Arg and Asp154-->Glu mutant enzymes were only moderately impaired in respect of catalysis. The results strongly indicate that Arg236, which is sandwiched between Arg176 and Lys239, would initiate the reaction upon enzyme-substrate interaction, through the abstraction of the proton at C5 of the galacturonopyranose ring. The positively charged residues Arg176 and Lys239 are responsible for lowering the p K a of Arg236. Arg176 and Lys239 are maintained in a charged state by interacting with Asp154 or bulk solvent respectively. The deprotonation of the Asp186-Asp221 pair was proposed to be responsible for a pH-driven conformational change of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677-689]. Substitution of Asp186 and Asp221 by Asn186 and Asn221 was expected to stabilize the enzyme. However, the Asp186-->Asn/Asp221-->Asn enzyme appeared less stable than the wild-type enzyme, even at pH 6.0, as evidenced by fluorescence studies. This demonstrates that the pH-dependent conformational change is not driven by deprotonation of the Asp186-Asp221 pair.
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PMID:Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A. 1241 64

The potato leafhopper, Empoasca fabae, is a pest of economic crops in the United States and Canada, where it causes damage known as hopperburn. Saliva, along with mechanical injury, leads to decreases in gas exchange rates, stunting and chlorosis. Although E. fabae saliva is known to induce plant responses, little knowledge exists of saliva composition at the molecular level. We subjected the salivary glands of E. fabae to Roche 454-pyrosequencing which resulted significant number (30,893) of expressed sequence tags including 2805 contigs and 28,088 singletons. A high number of sequences (78%) showed similarity to other insect species in GenBank, including Triboliumcastaneum, Drosophilamelanogaster and Acrythosiphonpisum. KEGG analysis predicted the presence of pathways for purine and thiamine metabolic, biosynthesis of secondary metabolites, drug metabolism, and lysine degradation. Pfam analysis showed a high number of cellulase and carboxylesterase protein domains. Expression analysis of candidate genes (alpha amylase, lipase, pectin lyase, etc.) among different tissues revealed tissue-specific expression of digestive enzymes in E. fabae. This is the first study to characterize the sialotranscriptome of E. fabae and the first for any species in the family of Cicadellidae. Due to the status of these insects as economic pests, knowledge of which genes are active in the salivary glands is important for understanding their impact on host plants.
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PMID:Transcriptome analysis of the salivary glands of potato leafhopper, Empoasca fabae. 2306