NMR solution structure of Apis mellifera chymotrypsin/cathepsin G inhibitor-1 (AMCI-1): structural similarity with Ascaris protease inhibitors.
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The three-dimensional structure of the 56 residue polypeptide Apis mellifera chymotrypsin/cathepsin G inhibitor 1 (AMCI-1) isolated from honey bee hemolymph was calculated based on 730 experimental NMR restraints. It consists of two approximately perpendicular beta-sheets, several turns, and a long exposed loop that includes the protease binding site. The lack of extensive secondary structure features or hydrophobic core is compensated by the presence of five disulfide bridges that stabilize both the protein scaffold and the binding loop segment. A detailed analysis of the protease binding loop conformation reveals that it is similar to those found in other canonical serine protease inhibitors. The AMCI-1 structure exhibits a common fold with a novel family of inhibitors from the intestinal parasitic worm Ascaris suum. The pH-induced conformational changes in the binding loop region observed in the Ascaris inhibitor ATI are absent in AMCI-1. Similar binding site sequences and structures strongly suggest that the lack of the conformational change can be attributed to a Glu-->Gln substitution at the P1' position in AMCI-1, compared to ATI. Analysis of amide proton temperature coefficients shows very good correlation with the presence of hydrogen bond donors in the calculated AMCI-1 structure. (+info)
Pheromone-regulated expression of sex pheromone plasmid pAD1-encoded aggregation substance depends on at least six upstream genes and a cis-acting, orientation-dependent factor.
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Conjugative transfer of Enterococcus faecalis-specific sex pheromone plasmids relies on an adhesin, called aggregation substance, to confer a tight cell-to-cell contact between the mating partners. To analyze the dependence of pAD1-encoded aggregation substance, Asa1, on pheromone induction, a variety of upstream fragments were fused to an alpha-amylase reporter gene, amyL, by use of a novel promoter probe vector, pAMY-em1. For pheromone-regulated alpha-amylase activity, a total of at least six genes, traB, traC, traA, traE1, orfY, and orf1, are required: TraB efficiently represses asa1 (by a mechanism unrelated to its presumptive function in pheromone shutdown, since a complete shutdown is observed exclusively in the presence of traC); only traC can relieve traB-mediated repression in a pheromone-dependent manner. In addition to traB, traA is required but not sufficient for negative control. Mutational inactivation of traE1, orfY, or orf1, respectively, results in a total loss of alpha-amylase activity for constructs normally mediating constitutive expression. Inversion of a fragment covering traA, P(0), and traE1 without disrupting any gene or control element switches off amyL or asa1 expression, indicating the involvement of a cis-acting, orientation-dependent factor (as had been shown for plasmid pCF10). Unexpectedly, pAD1 represses all pAMY-em1 derivatives in trans, while its own pheromone-dependent functions are unaffected. The discrepancy between the new data and those of former studies defining TraE1 as a trans-acting positive regulator is discussed. (+info)
D-Alanine substitution of teichoic acids as a modulator of protein folding and stability at the cytoplasmic membrane/cell wall interface of Bacillus subtilis.
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The extracytoplasmic folding of secreted proteins in Gram-positive bacteria is influenced by the microenvironment of the compartment into which they are translocated, namely the negatively charged matrix of the cell wall polymers. In this compartment, the PrsA lipoprotein facilitates correct post-translocational folding or prevents misfolding of secreted proteins. In this study, a secretion mutant of B. subtilis (prsA3) encoding a defective PrsA protein was mutagenized and screened for restored secretion of the AmyQ alpha-amylase. One mini-Tn10 insertion, which partially suppressed the secretion deficiency, was found to interrupt dlt, the operon involved in the d-alanylation of teichoic acids. The inactivation of dlt rescued the mutant PrsA3 protein from degradation, and the increased amount of PrsA3 was shown to enhance the secretion of PrsA-dependent proteins. Heterologous or abnormal secreted proteins, which are prone to degradation after translocation, were also stabilized and secreted in increased quantities from a dlt prsA(+) strain. Furthermore, the dlt mutation partially suppressed the lethal effect of PrsA depletion, suggesting that the dlt deficiency also leads to stabilization of an essential cell wall protein(s). Our results suggest that main influence of the increased net negative charge of the wall caused by the absence of d-alanine is to increase the rate of post-translocational folding of exported proteins. (+info)
A chimera-like alpha-amylase inhibitor suggesting the evolution of Phaseolus vulgaris alpha-amylase inhibitor.
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White kidney bean (Phaseolus vulgaris) contains two kinds of alpha-amylase inhibitors, one heat-stable (alpha AI-s) and one heat-labile (alpha AI-u). alpha AI-s has recently been revealed to be a tetrameric complex, alpha(2)beta(2), with two active sites [Kasahara et al. (1996) J. Biochem. 120, 177-183]. The present study was undertaken to reveal the molecular features of alpha AI-u, which is composed of three kinds of subunits, alpha, beta, and gamma. The gamma-subunit, in contrast to the alpha- and beta-subunits that are indistinguishable from the alpha- and beta-subunits of alpha AI-s, was found to correspond to a subunit of an alpha-amylase inhibitor-like protein, which has been identified as an inactive, evolutionary intermediate between arcelin and the alpha-amylase inhibitor in a P. vulgaris defense protein family. The polypeptide molecular weight of alpha AI-u determined by the light-scattering technique, together with the polypeptide molecular weights of the subunits, suggests that alpha AI-u is a trimeric complex, alpha beta gamma. The inhibition of alpha AI-u by increasing amounts of porcine pancreatic alpha-amylase (PPA) indicates that an inactive 1:1 complex is formed between alpha AI-u and PPA. Molecular weight estimation of the complex by the light-scattering technique confirmed that it is a complex of alpha AI-u with one PPA molecule. Thus it seems probable that alpha AI-u is an evolutionary intermediate of the P. vulgaris alpha-amylase inhibitor. (+info)
Introduction of raw starch-binding domains into Bacillus subtilis alpha-amylase by fusion with the starch-binding domain of Bacillus cyclomaltodextrin glucanotransferase.
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We constructed two types of chimeric enzymes, Ch1 Amy and Ch2 Amy. Ch1 Amy consisted of a catalytic domain of Bacillus subtilis X-23 alpha-amylase (Ba-S) and the raw starch-binding domain (domain E) of Bacillus A2-5a cyclomaltodextrin glucanotransferase (A2-5a CGT). Ch2 Amy consisted of Ba-S and D (function unknown) plus E domains of A2-5a CGT. Ch1 Amy acquired raw starch-binding and -digesting abilities which were not present in the catalytic part (Ba-S). Furthermore, the specific activity of Ch1 Amy was almost identical when enzyme activity was evaluated on a molar basis. Although Ch2 Amy exhibited even higher raw starch-binding and -digesting abilities than Ch1 Amy, the specific activity was lower than that of Ba-S. We did not detect any differences in other enzymatic characteristics (amylolytic pattern, transglycosylation ability, effects of pH, and temperature on stability and activity) among Ba-S, Ch1 Amy, and Ch2 Amy. (+info)
Alpha-amylase inhibitors from roselle (Hibiscus sabdariffa Linn.) tea.
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A roselle (Hibiscus sabdariffa Linn.) tea extract was found to have high inhibitory activity against porcine pancreatic alpha-amylase. Hibiscus acid and its 6-methyl ester were respectively isolated as active principles from the 50% methanol and acetone extracts of roselle tea. The activity of each isolate was compared to that of structurally related citric acid, a previously known inhibitor of fungal alpha-amylase. (+info)
Glucose and disaccharide-sensing mechanisms modulate the expression of alpha-amylase in barley embryos.
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The aim of this study was to investigate the sugar-sensing processes modulating the expression of alpha-amylase in barley (Hordeum vulgaris L. var Himalaya) embryos. The results highlight the existence of independent glucose (Glc) and disaccharides sensing. Glc treatment destabilizes the alpha-amylase mRNA. Non-metabolizable disaccharides repress alpha-amylase induction, but have no effects on transcript stability. Structure-function analysis indicates that a fructose (Fru) moiety is needed for disaccharide sensing. Lactulose (beta-galactose [Gal][1-->4]Fru), palatinose (Glc[1-->6]Fru), and turanose (Glc[1-->3]Fru) are not metabolized but repress alpha-amylase. Disrupting the fructosyl moiety of lactulose and palatinose, or replacing the Fru moiety of beta-Gal[1-->4]Fru with Glc or Gal results in molecules unable to repress alpha-amylase. Comparison of the molecular requirements for sucrose transport with those for disaccharide sensing suggests that these sugars are perceived possibly at the plasma membrane level independently from sucrose transport. (+info)
Inhibition of enzymic digestion of amylose by free fatty acids in vitro contributes to resistant starch formation.
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The effect of lipids on the enzymic breakdown of starch was investigated using an in vitro assay system. Mixtures of potato amylose, amylopectin and starch and various lipids were incubated at 37 degrees C for 10 min and subjected to digestion by alpha-amylase (EC 3.2.1.1) and amyloglucosidase (EC 3.2.1.33). Lauric, myristic, palmitic and oleic acids and lysolecithin inhibited enzymic hydrolysis of amylose by approximately 35% (P < 0.05). Stearic acid and cholesterol had no effect on the enzymic breakdown of amylose. Retrograded amylose was hydrolyzed less readily (P < 0.05) than solubilized amylose, but the breakdown was not further inhibited in the presence of lauric acid. Fatty acids had no effect on the enzymic hydrolysis of amylopectin, whereas inhibition by fatty acids of the breakdown of whole starch was consistent with only the amylose fraction being affected. The possibility that interactions between starch and fatty acids in the digestive tract could contribute to the formation of resistant starch is considered. (+info)