Inhibition of plant-pathogenic fungi by a corn trypsin inhibitor overexpressed in Escherichia coli.
The cDNA of a 14-kDa trypsin inhibitor (TI) from corn was subcloned into an Escherichia coli overexpression vector. The overexpressed TI was purified based on its insolubility in urea and then refolded into the active form in vitro. This recombinant TI inhibited both conidium germination and hyphal growth of all nine plant pathogenic fungi studied, including Aspergillus flavus, Aspergillus parasiticus, and Fusarium moniliforme. The calculated 50% inhibitory concentration of TI for conidium germination ranged from 70 to more than 300 microgram/ml, and that for fungal growth ranged from 33 to 124 microgram/ml depending on the fungal species. It also inhibited A. flavus and F. moniliforme simultaneously when they were tested together. The results suggest that the corn 14-kDa TI may function in host resistance against a variety of fungal pathogens of crops. (+info
Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system.
Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The A. oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A. oryzae glaA encoding glucoamylase and the agdA encoding alpha-glucosidase. To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A. nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter. Introduction of the sequence between -290 to -233 (the number indicates the distance in base pairs from the translation initiation point (+1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose. The sequence between -377 to -290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III. The sequence between -233 to -181 containing Region II had no effect on the expression. These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression. (+info
Overexpression of BiP in tobacco alleviates endoplasmic reticulum stress.
To study the role of the lumenal binding protein (BiP) in the transport and secretion of proteins, we have produced plants with altered BiP levels. Transgenic plants overexpressing BiP showed dramatically increased BiP mRNA levels but only a modest increase in BiP protein levels. The presence of degradation products in BiP overproducers suggests a regulatory mechanism that increases protein turnover when BiP is abundant. Antisense inhibition of BiP synthesis was not successful, demonstrating that even a minor reduction in the basal BiP level is deleterious to cell viability. Overexpression of BiP leads to downregulation of the basal transcript levels of endogenous BiP genes and greatly reduces the unfolded protein response. The data confirm that BiP transcription is regulated via a feedback mechanism that involves monitoring of BiP protein levels. To test BiP activity in vivo, we designed a functional assay, using the secretory protein alpha-amylase and a cytosolic enzyme as a control for cell viability. During tunicamycin treatment, an overall reduction of alpha-amylase synthesis was observed when compared with the cytosolic marker. We show that the tunicamycin effect is due to the depletion of BiP in the endoplasmic reticulum because coexpressed BiP alone is able to restore efficient alpha-amylase synthesis. This is a novel assay to monitor BiP activity in promoting secretory protein synthesis in vivo. (+info
Differential dependence of levansucrase and alpha-amylase secretion on SecA (Div) during the exponential phase of growth of Bacillus subtilis.
SecA, the translocation ATPase of the preprotein translocase, accounts for 0.25% of the total protein in a degU32(Hy) Bacillus subtilis strain in logarithmic phase. The SecA level remained constant irrespective of the demand for exoprotein production but dropped about 12-fold during the late stationary phase. Modulation of the level of functional SecA during the exponential phase of growth affected differently the secretion of levansucrase and alpha-amylase overexpressed under the control of the sacB leader region. The level of SecA was reduced in the presence of sodium azide and in the div341 thermosensitive mutant at nonpermissive temperatures. Overproduction of SecA was obtained with a multicopy plasmid bearing secA. The gradual decrease of the SecA level reduced the yield of secreted levansucrase with a concomitant accumulation of unprocessed precursor in the cells, while an increase in the SecA level resulted in an elevation of the production of exocellular levansucrase. In contrast, alpha-amylase secretion was almost unaffected by high concentrations of sodium azide or by very low levels of SecA. Secretion defects were apparent only under conditions of strong SecA deprivation of the cell. These data demonstrate that the alpha-amylase and levansucrase precursors markedly differ in their dependency on SecA for secretion. It is suggested that these precursors differ in their binding affinities for SecA. (+info
Genetic regulation of tissue-specific expression of amylase structural genes in Drosophila melanogaster.
Laboratory strains of Drosophila melanogaster were screened for spatial variations in adult midgut alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 22.214.171.124) expression. No strain-specific differences were found anteriorly, but three patterns of activity were discerned in the posterior midgut: A, activity throughout most of the region; B, activity in the anterior part of the region; and C, little or no activity. Alleles of a control gene, map, are responsible for this tissue-specific regulation of activity; e.g., mapA homozygotes produce the A pattern and mapC homozygotes the C pattern. The map locus was placed at 2--80 +/- on the genetic map of chromosome 2R, about two crossover units distal to the Amy structural gene region for alpha-amylase. Electrophoretic studies showed that mapA is trans acting in mapA/mapC flies, allowing expression of amylase isozymes coded for by genes on the opposite chromosome. The map gene behaves as a temporal gene that is clearly separable from the tightly linked, duplicated Amy structural genes. (+info
Molecular cloning and primary structure analysis of porcine pancreatic alpha-amylase.
A cDNA library was constructed in a Uni-ZAP XR vector using mRNA isolated from porcine pancreas. A full-length alpha-amylase cDNA was obtained using a combination of library screening and nested polymerase chain reaction. Sequencing of the clone revealed a 1536-nucleotide (nt) open reading frame encoding a protein of 496 amino acid (aa) residues with a signal peptide of 15 aa. The calculated molecular mass of the enzyme was 55354 Da, in accordance with those of the purified porcine pancreatic alpha-amylase forms (PPAI and PPAII) as determined by mass spectrometry. A comparison of the deduced aa sequence with published peptidic sequences of PPAI identified a number of mismatches. The sequence of the cDNA reported here provides a sequence reference for PPA in excellent agreement with the refined three-dimensional structures of both PPAI and PPAII. No evidence for a second variant was found in the cDNA library and it is most likely that PPAI and PPAII are two forms of the same protein. The primary structure of PPA shows high homology with human, mouse and rat pancreatic alpha-amylases. The 304-310 region, corresponding to a mobile loop involved in substrate binding and processing near the active site, is fully conserved. (+info
Cloning, mutagenesis, and structural analysis of human pancreatic alpha-amylase expressed in Pichia pastoris.
Human pancreatic alpha-amylase (HPA) was expressed in the methylotrophic yeast Pichia pastoris and two mutants (D197A and D197N) of a completely conserved active site carboxylic acid were generated. All recombinant proteins were shown by electrospray ionization mass spectrometry (ESI-MS) to be glycosylated and the site of attachment was shown to be Asn461 by peptide mapping in conjunction with ESI-MS. Treatment of these proteins with endoglycosidase F demonstrated that they contained a single N-linked oligosaccharide and yielded a protein product with a single N-acetyl glucosamine (GlcNAc), which could be crystallized. Solution of the crystal structure to a resolution of 2.0 A confirmed the location of the glycosyl group as Asn461 and showed that the recombinant protein had essentially the same conformation as the native enzyme. The kinetic parameters of the glycosylated and deglycosylated wild-type proteins were the same while the k(cat)/Km values for D197A and D197N were 10(6)-10(7) times lower than the wild-type enzyme. The decreased k(cat)/Km values for the mutants confirm that D197 plays a crucial role in the hydrolytic activity of HPA, presumably as the catalytic nucleophile. (+info
Chemical modification of lysine side chains of cyclodextrin glycosyltransferase from Thermoanaerobacter causes a shift from cyclodextrin glycosyltransferase to alpha-amylase specificity.
Cyclodextrin glycosyltransferases and alpha-amylases are two groups of enzymes with related secondary structures. However, cyclodextrin glycosyltransferases display transferase activities not present in alpha-amylases, probably derived from the existence of two more domains and different amino acid sequences. The hydrolytic activity of cyclodextrin glycosyltransferases is generally quite low, except for two cyclodextrin glycosyltransferases from termophiles. In this work, we have carried out the chemical modification (with acetic anhydride) of the amino groups of cyclodextrin glycosyltransferase from Thermoanaerobacter to assess their contributions to protein function. The acetylated cyclodextrin glycosyltransferase showed a significant reduction of its cyclization, coupling and disproportionation activities. Surprisingly, the hydrolytic (saccharifying) activity was slightly enhanced. These results suggest the participation of one or more lysine side chains in the interactions contributing to the transferase activity, either in any of the S11 subsites or in the acceptor binding site. (+info