Yeast glucoamylases: molecular-genetic and structural characterization

Biologia ◽  
2010 ◽  
Vol 65 (4) ◽  
Author(s):  
Eva Hostinová ◽  
Juraj Gašperík
Keyword(s):  
Filamentous Fungi ◽  
Tertiary Structure ◽  
Biochemical Characterization ◽  
Recombinant Expression ◽  
Molecular Genetic ◽  
Extracellular Enzyme ◽  
Major Application ◽  
The Given

AbstractGlucoamylase is an extracellular enzyme produced mainly by microorganisms. It belongs to the commercially frequently exploited biocatalysts. The major application of glucoamylase is in the starch bioprocessing to produce glucose and in alcoholic fermentations of starchy materials. Filamentous fungi have been the source of glucoamylases for industrial purposes as well as an object of numerous research studies. Some yeasts also secrete a large amount of glucoamylase with biochemical characteristics slightly different from those of filamentous fungi. Modern biotechnological applications require glucoamylases of certain properties optimal for a given process. Novel biocatalysts can be prepared from already existing enzymes using techniques of protein engineering or directed evolution. Tailoring of a commercial glucoamylase requires knowledge, on a molecular level, of structure/function relationships of enzymes originating from various sources and having different catalytic properties. Sequences of the cloned genes, their recombinant expression and the tertiary structure determination of glucoamylase are prerequisite to obtain such information. The presented review focuses on molecular-genetic and structural aspects of yeast glucoamylases, supplemented with the basic biochemical characterization of the given enzymes.

scholarly journals Structure and Synthesis of Antifungal Disulfide β-Strand Proteins from Filamentous Fungi

2018 ◽  
Vol 7 (1) ◽  
pp. 5 ◽  
Author(s):  
Györgyi Váradi ◽  
Gábor Tóth ◽  
Gyula Batta
Keyword(s):  
Filamentous Fungi ◽  
Disulfide Bonds ◽  
Antimicrobial Agents ◽  
Tertiary Structure ◽  
Fungal Infections ◽  
Recombinant Expression ◽  
Antifungal Protein ◽  
Significant Group ◽  
Antifungal Proteins ◽  
Neosartorya Fischeri

The discovery and understanding of the mode of action of new antimicrobial agents is extremely urgent, since fungal infections cause 1.5 million deaths annually. Antifungal peptides and proteins represent a significant group of compounds that are able to kill pathogenic fungi. Based on phylogenetic analyses the ascomycetous, cysteine-rich antifungal proteins can be divided into three different groups: Penicillium chrysogenum antifungal protein (PAF), Neosartorya fischeri antifungal protein 2 (NFAP2) and “bubble-proteins” (BP) produced, for example, by P. brevicompactum. They all dominantly have β-strand secondary structures that are stabilized by several disulfide bonds. The PAF group (AFP antifungal protein from Aspergillus giganteus, PAF and PAFB from P. chrysogenum, Neosartorya fischeri antifungal protein (NFAP)) is the best characterized with their common β-barrel tertiary structure. These proteins and variants can efficiently be obtained either from fungi production or by recombinant expression. However, chemical synthesis may be a complementary aid for preparing unusual modifications, e.g., the incorporation of non-coded amino acids, fluorophores, or even unnatural disulfide bonds. Synthetic variants up to ca. 6–7 kDa can also be put to good use for corroborating structure determination. A short overview of the structural peculiarities of antifungal β-strand disulfide bridged proteins will be given. Here, we describe the structural propensities of some known antifungal proteins from filamentous fungi which can also be prepared with modern synthetic chemistry methods.

scholarly journals Recombinant expression, purification and biochemical characterization of kievitone hydratase from Nectria haematococca

PLoS ONE ◽  
2018 ◽  
Vol 13 (2) ◽  
pp. e0192653 ◽  
Author(s):  
Matthias Engleder ◽  
Melissa Horvat ◽  
Anita Emmerstorfer-Augustin ◽  
Tamara Wriessnegger ◽  
Stefanie Gabriel ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Recombinant Expression ◽  
Nectria Haematococca

scholarly journals Cloning, recombinant expression and biochemical characterization of the murine CD83 molecule which is specifically upregulated during dendritic cell maturation

FEBS Letters ◽  
1999 ◽  
Vol 461 (3) ◽  
pp. 211-216 ◽  
Author(s):  
Susanne Berchtold ◽  
Petra Mühl-Zürbes ◽  
Christine Heufler ◽  
Patrizia Winklehner ◽  
Gerold Schuler ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Biochemical Characterization ◽  
Recombinant Expression ◽  
Dendritic Cell Maturation ◽  
Cell Maturation

scholarly journals Genetic and Biochemical Characterization of a Novel Monoterpene ɛ-Lactone Hydrolase from Rhodococcus erythropolis DCL14

2001 ◽  
Vol 67 (2) ◽  
pp. 733-741 ◽  
Author(s):  
Cécile J. B van der Vlugt-Bergmans ◽  
Mariët J. van der Werf
Keyword(s):  
Ring Opening ◽  
Biochemical Characterization ◽  
Rhodococcus Erythropolis ◽  
Open Reading Frames ◽  
Apparent Homogeneity ◽  
Terminal Amino ◽  
Acyl Coenzyme A ◽  
Reading Frames

ABSTRACT A monoterpene ɛ-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30°C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB(monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.

Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Biochemical Features and Therapeutic Approaches

2007 ◽  
Vol 27 (1-3) ◽  
pp. 151-163 ◽  
Author(s):  
M. C. Lara ◽  
M. L. Valentino ◽  
J. Torres-Torronteras ◽  
M. Hirano ◽  
R. Martí
Keyword(s):  
Molecular Genetic ◽  
In Vivo Studies ◽  
Gene Encoding ◽  
Mitochondrial Neurogastrointestinal Encephalomyopathy ◽  
Mtdna Replication ◽  
Biochemical Features

Over the last 15 years, important research has expanded our knowledge of the clinical, molecular genetic, and biochemical features of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). The characterization of mitochondrial involvement in this disorder and the seminal determination of its genetic cause, have opened new possibilities for more detailed and deeper studies on the pathomechanisms in this progressive and fatal disease. It has been established that MNGIE is caused by mutations in the gene encoding thymidine phosphorylase (TP), which lead to absolute or nearly complete loss of its catalytic activity, producing systemic accumulations of its substrates, thymidine (dThd) and deoxyuridine (dUrd). Findings obtained from in vitro and in vivo studies indicate that the biochemical imbalances specifically impair mitochondrial DNA (mtDNA) replication, repair, or both leading to mitochondrial dysfunction. We have proposed that therapy for MNGIE should be aimed at reducing the concentrations of these toxic nucleosides to normal or nearly normal levels. The first treatment, allogeneic stem-cell transplantation (alloSCT) reported in 2006, produced a nearly full biochemical correction of the dThd and dUrd imbalances in blood. Clinical follow-up of this and other patients receiving alloSCT is necessary to determine whether this and other therapies based on a permanent restoration of TP will be effective treatment for MNGIE.

scholarly journals Structural characterization of UDP-galactopyranose mutase from eukaryotic pathogens

2013 ◽  
Author(s):  
◽  
Richa Dhatwalia
Keyword(s):  
Drug Target ◽  
Pathogenic Bacteria ◽  
Leishmania Major ◽  
Biochemical Characterization ◽  
Three Dimensional ◽  
Protozoan Parasites ◽  
Enzymatic Mechanism ◽  
Structural Details

UDP-galactopyranose mutase (UGM) is a unique flavoenzyme that catalyzes the interconversion between UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf), without any net transfer of electrons. UGM is a central enzyme involved in the biosynthesis of galactofuranose (Galf). Galf forms a major component of different glycoconjugate structures, lipids and polysaccharides of disease-causing fungi, Aspergillus fumigatus and protozoan parasites such as Trypanosoma cruzi and Leishmania major. Current treatments for diseases caused by these pathogens are limited and use compounds that are either highly toxic or expensive. Thus, new drug development strategies are required for combating these lethal diseases. The unique chemistry of UGMs and its implication in the virulence of pathogenic bacteria, fungi and protozoa and its absence in humans make it a potential drug target. Though bacterial UGMs have been somewhat characterized in detail using structural and biochemical methods, major questions about the catalytic and structural properties of eukaryotic UGMs remain unanswered. Thus, the determination of three-dimensional structures of eukaryotic UGMs might help us in elucidating the enzymatic mechanism of this class of enzymes and potential inhibitor design. The research described in this dissertation address these longstanding questions by providing the first three-dimensional structural details and biochemical characterization of eukaryotic UGMs.

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