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1.
Int J Biol Macromol ; 39(4-5): 286-90, 2006 Nov 15.
Article in English | MEDLINE | ID: mdl-16797066

ABSTRACT

Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 x 10(-6) M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl-alpha-d-mannopyranoside.


Subject(s)
Concanavalin A/metabolism , Glucan 1,4-alpha-Glucosidase/metabolism , Aspergillus niger/enzymology , Binding Sites , Chemical Precipitation , Concanavalin A/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Glucan 1,4-alpha-Glucosidase/antagonists & inhibitors , Glucan 1,4-alpha-Glucosidase/chemistry , Glycosylation , Kinetics , Methylmannosides/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomycopsis/enzymology , Surface Plasmon Resonance
2.
Mol Cell Biol ; 21(15): 5179-89, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11438672

ABSTRACT

p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (K(d) 0.6 microM) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (K(d) 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.


Subject(s)
Protein Serine-Threonine Kinases/metabolism , Animals , Catalysis , Catalytic Domain , Chromatography, Gel , Circular Dichroism , Dimerization , Dose-Response Relationship, Drug , Enzyme Activation , GTP-Binding Proteins/metabolism , Glutathione Transferase/metabolism , Kinetics , Models, Biological , Phosphorylation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/metabolism , Signal Transduction , Spectrometry, Fluorescence , Time Factors , cdc42 GTP-Binding Protein/metabolism , p21-Activated Kinases , rac GTP-Binding Proteins/metabolism
3.
Eur J Biochem ; 264(3): 756-64, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10491121

ABSTRACT

The mutation Gly467-->Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the kcat towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (Ka congruent with 10(13).M(-1)) than the Glu enzyme (Ka congruent with 10(12).M(-1)). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (Ka congruent with 10(4).M(-1)) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.


Subject(s)
Genes, Fungal , Glucan 1,4-alpha-Glucosidase/chemistry , Glucan 1,4-alpha-Glucosidase/genetics , Saccharomycopsis/enzymology , Saccharomycopsis/genetics , Acarbose , Base Sequence , Calorimetry , Carbohydrate Sequence , Catalytic Domain/genetics , DNA Primers/genetics , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Stability , Genetic Variation , Glucan 1,4-alpha-Glucosidase/metabolism , Hydrogen Bonding , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Point Mutation , Substrate Specificity , Thermodynamics , Trisaccharides/chemistry , Trisaccharides/pharmacology
6.
Acta Crystallogr D Biol Crystallogr ; 54(Pt 5): 854-66, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-9757101

ABSTRACT

The yeast Saccharomycopsis fibuligera produces a glucoamylase which belongs to sequence family 15 of glycosyl hydrolases. The structure of the non-glycosyl-ated recombinant enzyme has been determined by molecular replacement and refined against 1.7 A resolution synchrotron data to an R factor of 14.6%. This is the first report of the three-dimensional structure of a yeast family 15 glucoamylase. The refinement from the initial molecular-replacement model was not straightforward. It involved the use of an unrestrained automated refinement procedure (uARP) in combination with the maximum-likelihood refinement program REFMAC. The enzyme consists of 492 amino-acid residues and has 14 alpha-helices, 12 of which form an (alpha/alpha)6 barrel. It contains a single catalytic domain but no starch-binding domain. The fold of the molecule and the active site are compared to the known structure of the catalytic domain of a fungal family 15 glucoamylase and are shown to be closely similar. The active- and specificity-site residues are especially highly conserved. The model of the acarbose inhibitor from the analysis of the fungal enzyme fits tightly into the present structure. The active-site topology is a pocket and hydrolysis proceeds with inversion of the configuration at the anomeric carbon. The enzyme acts as an exo-glycosyl hydrolase. There is a Tris [2-amino-2-(hydroxymethyl)-1,3-propanediol] molecule acting as an inhibitor in the active-site pocket.


Subject(s)
Fungal Proteins/chemistry , Glucan 1,4-alpha-Glucosidase/chemistry , Protein Conformation , Yeasts/enzymology , Amino Acid Sequence , Binding Sites , Crystallization , Crystallography, X-Ray , Glycoside Hydrolases/chemistry , Models, Molecular , Molecular Sequence Data , Protein Folding , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity
7.
Acta Crystallogr D Biol Crystallogr ; 53(Pt 6): 782-3, 1997 Nov 01.
Article in English | MEDLINE | ID: mdl-15299869

ABSTRACT

The active non-glycosylated glucoamylase, overexpressed from the Saccharomycopsis fibuligera GLU1 gene in Escherichia coli BL21(DE3), has been purified from the solubilized inclusion bodies and then renatured in vitro. Crystals of the recombinant glucoamylase were obtained by vapour diffusion using PEG as precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell dimensions of a = 58.1, b = 87.8 and c = 99.9 A, and diffract to 1.7 A resolution. This is the first report of the crystallization of the full-length glucoamylase corresponding to the mature enzyme.

8.
Biochem Biophys Res Commun ; 224(3): 790-5, 1996 Jul 25.
Article in English | MEDLINE | ID: mdl-8713124

ABSTRACT

The truncated GLA1 gene encoding the mature form of glucoamylase from the yeast Saccharomycopsis fibuligera has been over-expressed in Escherichia coli using the IPTG inducible pET system. Over-expression has led to the accumulation of insoluble glucoamylase in inclusion bodies from which an electrophoretically homogeneous active enzyme has been prepared yielding 30 mg per litre medium. This protein represents an N-terminus Met-free, non-glycosylated product which displays the identical specific activity of 45 units/mg and reduced thermal stability when compared to glycosylated enzymes isolated from Saccharomyces cerevisiae carrying the GLA1 gene. These data suggest that S. cerevisiae glycosylation of S. fibuligera glucoamylase does not play a critical role in enzymatic activity but that it does contribute to its thermal stability.


Subject(s)
Glucan 1,4-alpha-Glucosidase/genetics , Saccharomycopsis/enzymology , Base Sequence , Crystallography, X-Ray , DNA Primers , Escherichia coli/genetics , Glucan 1,4-alpha-Glucosidase/chemistry , Glucan 1,4-alpha-Glucosidase/metabolism , Glycosylation , Hydrogen-Ion Concentration , Molecular Sequence Data , Protein Denaturation , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship
9.
FEMS Microbiol Lett ; 67(1): 103-8, 1991 Sep 15.
Article in English | MEDLINE | ID: mdl-1840532

ABSTRACT

The nucleotide sequence of the 2544-bp PstI fragment carrying the glucoamylase gene of Saccharomycopsis fibuligera KZ, designated as GLA1, has been determined. When compared with the nucleotide sequence of the GLU1 gene one nucleotide substitution was found in the 321- bp of the 5'-flanking region: 24 nucleotides were altered within the 1557 bp of the structural gene causing the deduced protein products of both genes to differ in three amino acids in the signal-peptide region and in eight amino acids of the mature protein. Six nucleotide insertions and 27 substitutions were in the 663 bp of the 3'-flanking region. The gene product expressed and secreted in Saccharomyces cerevisiae into the functional enzyme was not homogeneous. In situ detection of the enzyme in a polyacrylamide gel revealed two dominant and three minor bands.


Subject(s)
Genes, Fungal , Glucan 1,4-alpha-Glucosidase/genetics , Saccharomycopsis/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular/methods , DNA, Bacterial/genetics , Escherichia coli/genetics , Molecular Sequence Data , Plasmids , Protein Sorting Signals/genetics , Restriction Mapping , Saccharomycopsis/enzymology
10.
J Basic Microbiol ; 30(4): 301-4, 1990.
Article in English | MEDLINE | ID: mdl-2200870

ABSTRACT

A Sau3AI digest of total DNA from Saccharomycopsis fibuligera was cloned with the yeast-integrating plasmid YEp24 delta EcoRI and the capacity for autonomous replication (ARS) was assayed in yeast. From eighty clones, five mitoticaly unstable yeast transformants were picked up, recombinant plasmids from these clones were recovered with Escherichia coli, mapped, hybridized with total DNA of S. fibuligera and tested to mitotical stability in Saccharomyces cerevisiae. Experiments suggested the existence of DNA sequences from dimorphic Saccharomycopsis fibuligera with ARS activity in Saccharomyces cerevisiae.


Subject(s)
DNA Replication , DNA, Fungal/analysis , Saccharomyces cerevisiae/genetics , Saccharomycetales/genetics , Saccharomycopsis/genetics , Base Sequence , Transformation, Genetic
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