Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.10.3.2 (laccase)
 4,656 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Acidification of vesicular compartments plays an important role in a number of cellular transport processes, including protein secretion, metal cofactor insertion, glycosylation and pH stability. In the present study, we identify and characterize a component of the vesicular proton pump, Vph1p, to determine its role in the virulence of the AIDS-related fungal pathogen Cryptococcus neoformans. Insertional mutagenesis and plasmid rescue were used to identify the VPH1 gene by screening for mutants defective in laccase activity. Disruption of VPH1 resulted in defects in three virulence factors (capsule production, laccase and urease expression), as well as a growth defect at 37 degrees C, but only a small growth reduction at 30 degrees C. These effects were duplicated by the vacuolar (H+)-ATPase inhibitor bafilomycin A1. Furthermore, the vph1 insertional mutant was also avirulent in a mouse meningo-encephalitis model. Complementation of the insertional mutant with wild-type VPH1 resulted in a recovery of virulence factor expression, normal growth at 37 degrees C and restoration of full virulence. These studies establish the importance of the VPH1 gene and vesicular acidification in the virulence of C. neoformans.
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PMID:Multiple virulence factors of Cryptococcus neoformans are dependent on VPH1. 1173 51

Previous studies have shown that a Deltavph1 Cryptococcus neoformans mutant defective in vesicular acidification lacked several important virulence factors including a copper-containing laccase and was avirulent in a mouse model. In the present studies, we characterized laccase transcription and protein production to obtain insights into the mechanism of the vph1 mutation in this pathogen. Although transcription and protein expression were somewhat reduced, laccase protein was found to be successfully translated and correctly targeted to the cell wall in the Deltavph1 mutant as shown by Western blot and immuno-electron microscopy, despite a complete lack of laccase activity. Laccase activity was substantially restored in metabolically active Deltavph1 cells at 30 degrees C by addition of 100 micro M copper sulphate. This restoration by copper was found to occur through both transcriptional and post-translational mechanisms. Laccase transcriptional induction by copper was found to be dependent on enhancer region II within the 5'-untranslated region of CNLAC1. Copper was also found to restore partial activity to Deltavph1 cells at 0 degrees C, suggesting that cell wall laccase was expressed in the mutant as an apo-enzyme. Apo-laccase restoration by copper was found to be facilitated by an acidic environment, consistent with a role for the vacuolar (H+)-ATPase proton pump in copper assembly of laccase in C. neoformans.
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PMID:Copper-mediated reversal of defective laccase in a Deltavph1 avirulent mutant of Cryptococcus neoformans. 1258 55

Here the identification and characterization of a gene encoding a copper-trafficking enzyme, ctaA (copper-transporting ATPase), from the basidiomycete Trametes versicolor are described. This P-type copper ATPase gene has two alleles, differing primarily in the length of the second, unusually long intron, and encodes a 983 aa protein with 40 % sequence identity to yeast Ccc2p. Overexpression of ctaA in yeast grown in the presence of copper led to a 15-fold increase in laccase yields, while overexpression of ctaA and tahA, a previously identified copper homeostasis gene of T. versicolor, was additive, leading to a 20-fold increase in laccase production. In T. versicolor, overexpression of ctaA and tahA led to an eightfold increase in laccase expression, and a cotransformant still expressed laccase at 3000 micro M copper when hardly any laccase activity is detected in the wild-type strain. Apparently, at low to moderate levels of copper tahA and ctaA overexpression disturbs the normal hierarchy of copper distribution, resulting in more being directed to the Golgi, while with high copper amounts that normally switch on the copper detoxification processes, tahA and ctaA gene products seem to out-compete the metallothionein copper chaperones, meaning laccase is still supplied with copper. These results may lead to a better understanding of copper trafficking and the hierarchy of copper distribution in the cell, and possibly be useful for constructing laccase-overproducing strains for biotechnological purposes.
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PMID:Identification and functional expression of ctaA, a P-type ATPase gene involved in copper trafficking in Trametes versicolor. 1290 44

The bacterium Ornithobacterium rhinotracheale has been recognized as an emerging pathogen in poultry since about 10 years ago. Knowledge of this bacterium and its mechanisms of virulence is still very limited. Here we report the development of a transformation system that enables genetic modification of O. rhinotracheale. The system is based on a cryptic plasmid, pOR1, that was derived from an O. rhinotracheale strain of serotype K. Sequencing indicated that the plasmid consisted of 14,787 nucleotides. Sequence analysis revealed one replication origin and several rep genes that control plasmid replication and copy number, respectively. In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase. Reverse transcription-PCR demonstrated that these genes were transcribed. Other putative open reading frames exhibited similarities with a virulence-associated protein in Actinobacillus actinomycetemcomitans and a number of genes coding for proteins with unknown function. An Escherichia coli-O. rhinotracheale shuttle plasmid (pOREC1) was constructed by cloning the replication origin and rep genes from pOR1 and the cfxA gene from Bacteroides vulgatus, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using E. coli as a host. pOREC1 was electroporated into O. rhinotracheale and yielded cefoxitin-resistant transformants. The pOREC1 isolated from these transformants was reintroduced into E. coli, demonstrating that pOREC1 acts as an independent replicon in both E. coli and O. rhinotracheale, fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains.
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PMID:Characterization of plasmid pOR1 from Ornithobacterium rhinotracheale and construction of a shuttle plasmid. 1546 24

Herein we propose a facile, versatile and selective chemo-enzymatic synthesis of substituted (E)-2,3-diaryl-5-styryl-trans-2,3-dihydrobenzofurans based on the exploitation of the laccase-mediated oxidative (homo)coupling of (E)-4-styrylphenols. Thanks to this novel synthetic strategy, a library of benzofuran-based potential allosteric activators of the Heat shock protein 90 (Hsp90) was easily prepared. Moreover, considering their structural analogies to previously reported allosteric modulators, the sixteen new compounds synthesized in this work were tested in vitro for their potential stimulatory action on the ATPase activity of the molecular chaperone Hsp90. Combining experimental and computational results, we propose a mechanism of action for these compounds, and expand the structure-activity relationship (SAR) information available for benzofuran-based Hsp90 activators.
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PMID:Chemo-enzymatic synthesis of (E)-2,3-diaryl-5-styryl-trans-2,3-dihydrobenzofuran-based scaffolds and their in vitro and in silico evaluation as a novel sub-family of potential allosteric modulators of the 90 kDa heat shock protein (Hsp90). 2972 82