Antisense silencing of the creA gene in Aspergillus nidulans.
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Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was approximately one-half of that achieved in a null creA mutant. Unlike results for that mutant, however, growth parameters and colony morphology in the antisense transformants were not affected. (+info)
Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system.
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A series of chimeric alpha-amylase genes derived from amyL, which encodes the liquefying alpha-amylase from Bacillus licheniformis, were constructed in vitro using gene splicing techniques. The gene constructs were cloned in Bacillus subtilis, where their ability to direct the synthesis and secretion of active alpha-amylase was determined. Detectable alpha-amylase activity was observed for some, but not all, of the chimeric proteins. Studies on the secretion of wild-type AmyL and its chimeric derivatives revealed that, whilst these proteins were stable in the extracellular milieu, all were subject to some degree of degradation during secretion. The chimeric enzymes were degraded to a greater extent than the native enzyme. These findings suggest that cell-associated proteolysis is a significant problem affecting the use of B. subtilis as host bacterium for the production of heterologous proteins. (+info)
Psychrophilic enzymes: revisiting the thermodynamic parameters of activation may explain local flexibility.
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Basic theoretical and practical aspects of activation parameters are briefly reviewed in the context of cold-adaptation. In order to reduce the error impact inherent to the transition state theory on the absolute values of the free energy (DeltaG(#)), enthalpy (DeltaH(#)) and entropy (DeltaS(#)) of activation, it is proposed to compare the variation of these parameters between psychrophilic and mesophilic enzymes, namely Delta(DeltaG(#))(p-m), Delta(DeltaH(#))(p-m) and Delta(DeltaS(#))(p-m). Calculation of these parameters from the available literature shows that the main adaptation of psychrophilic enzymes lies in a significant decrease of DeltaH(#), therefore leading to a higher k(cat), especially at low temperatures. Moreover, in all cases including cold-blooded animals, DeltaS(#) exerts an opposite and negative effect on the gain in k(cat). It is argued that the magnitude of this counter-effect of DeltaS(#) can be reduced by keeping some stable domains, while increasing the flexibility of the structures required to improve catalysis at low temperature, as demonstrated in several cold-active enzymes. This enthalpic-entropic balance provides a new approach explaining the two types of conformational stability detected by recent microcalorimetric experiments on psychrophilic enzymes. (+info)
Evolution of alpha-amylases: architectural features and key residues in the stabilization of the (beta/alpha)(8) scaffold.
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We provide a comprehensive analysis of the current enzymes with alpha-amylase activity (AAMYs) that belong to family 13 glycoside hydrolase (GH-13; 144 Archaea, Bacteria, and Eukaryota sequences from 87 different species). This study aims to further knowledge of the evolutionary molecular relationships among the sequences of their A and B domains with special emphasis on the correlation between what is observed in the structures and protein evolution. Multialignments for the A domain distinguish two clusters for sequences from Archaea organisms, eight for sequences from Bacteria organisms, and three for sequences from Eukaryota organisms. The clusters for Bacteria do not follow any strict taxonomic pathway; in fact, they are rather scattered. When we compared the A domains of sequences belonging to different kingdoms, we found that various pairs of clusters were significantly similar. Using either sequence similarity with crystallized structures or secondary-structure prediction methods, we identified in all AAMYs the eight putative beta-strands that constitute the beta-sheet in the TIM barrel of the A domain and studied the packing in its interior. We also discovered a "hidden homology" in the TIM barrel, an invariant Gly located upstream in the sequence before the conserved Asp in beta-strand 3. This Gly precedes an alpha-helix and is actively involved in capping its N-terminal end with a capping box. In all cases, a Schellman motif caps the C-terminal end of this helix. (+info)
Protein engineering of bacterial alpha-amylases.
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alpha-Amylases constitute a very diverse family of glycosyl hydrolases that cleave alpha1-->4 linkages in amylose and related polymers. Recent structural and mutagenic studies of archeael, mammalian and bacterial alpha-amylases have resulted in a wealth of information on the catalytic mechanism and on the structural features of this enzyme class. Because of their high thermo-stability, the Bacillus alpha-amylases have found widespread use in industrial processes, and much attention has been devoted to optimising these enzymes for the very harsh conditions encountered there. Stability has been a major area of focus in this respect, and several remarkably stable bacterial alpha-amylases have been produced by bioengineering techniques. Protein engineering studies of pH-activity profiles and of substrate specificities have also been initiated, although without much success. In the coming years it is likely, however, that the focus of alpha-amylase engineering will shift from engineering stability to these new areas. (+info)
Approaches for deciphering the structural basis of low temperature enzyme activity.
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An increasing number of enzymes active at low temperature are being studied to help determine the structural features important for cold-activity. This review examines the diversity of prokaryotic cold-active enzymes and the features proposed to account for low temperature activity. We then consider the difficulty of identifying the key structural features needed for cold-activity and the need to compare enzymes having different temperature optima from phylogenetically related organisms to determine features responsible for low temperature activity. In addition to studying naturally occurring enzymes, directed evolution experiments are discussed as methods for examining the proposed mechanisms influencing the thermal dependence of activity. (+info)
Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline.
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Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 microM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37 degrees C was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 microM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 microM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 +/- 0.8 microM and k cat = 48.4 +/- 1.0 min(-1). The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 +/- 10, 1,098 +/- 91, 38.6 +/- 5.2 and 37,340 +/- 5,400 microM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 +/- 92.8 and 310,500 +/- 38,600 microM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds. (+info)
Mechanism of porcine pancreatic alpha-amylase. Inhibition of amylose and maltopentaose hydrolysis by alpha-, beta- and gamma-cyclodextrins.
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The effects of alpha-, beta- and gamma-cyclodextrins on the amylose and maltopentaose hydrolysis catalysed by porcine pancreatic alpha-amylase (PPA) were investigated. The results of the statistical analysis performed on the kinetic data using the general initial velocity equation of a one-substrate reaction in the presence of one inhibitor indicate that the type of inhibition involved depends on the substrate used: the inhibition of amylose hydrolysis by alpha-, beta- and gamma-cyclodextrin is of the competitive type, while the inhibition of maltopentaose hydrolysis is of the mixed noncompetitive type. Consistently, the Lineweaver-Burk plots intersect on the vertical axis when amylose is used as the substrate, while in the case of maltopentaose, the intersection occurs at a point located in the second quadrant. The inhibition of the hydrolysis therefore involves only one abortive complex, PPA-cyclodextrin, when amylose is used as the substrate, while two abortive complexes, PPA-cyclodextrin and PPA-maltopentaose-cyclodextrin, are involved with maltopentaose. The mixed noncompetitive inhibition thus shows the existence of one accessory binding site. In any case, only one molecule of inhibitor binds to PPA. In line with these findings, the difference spectra of PPA produced by alpha-, beta- and gamma-cyclodextrin indicate that binding occurs at a tryptophan and a tyrosine residue. The corresponding dissociation constants and the inhibition constants obtained using the kinetic approach are in the same range (1.2-7 mM). The results obtained here on the inhibition of maltopentaose hydrolysis by cyclodextrin are similar to those previously obtained with acarbose as the inhibitor [Alkazaz, M., Desseaux, V., Marchis-Mouren, G., Prodanov, E. & Santimone, M. (1998) Eur. J. Biochem. 252, 100-107], but differ from those obtained with amylose as the substrate and acarbose as inhibitor [Alkazaz, M., Desseaux, V., Marchis-Mouren, G., Payan, F., Forest, E. & Santimone, M. (1996) Eur. J. Biochem. 241, 787-796]. It is concluded that the hydrolysis of both long and short chain substrates requires at least one secondary binding site, including a tryptophan residue. (+info)