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Stefan Janecek - One of the best experts on this subject based on the ideXlab platform.
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A novel GH13 subfamily of α-amylases with a pair of tryptophans in the helix α3 of the catalytic TIM-barrel, the LPDlx signature in the conserved sequence region V and a conserved aromatic motif at the C-terminus
Biologia, 2015Co-Authors: Stefan Janecek, Andrea Kuchtová, Soňa PetrovičováAbstract:The α-amylase Enzyme Specificity has been classified in the Carbohydrate-Active Enzyme (CAZy) database into the families GH13, GH57, GH119 and eventually also GH126. α-Amylase is a glycoside hydrolase (GH) that catalyses in an endo-fashion the hydrolysis of the α-1,4-glucosidic linkages in starch and related α-glucans employing the retaining reaction mechanism. The family GH13 is the main a-amylase family with more than 28,000 members and 30 different specificities. The entire family GH13 has already been divided into 40 subfamilies; the a-amylase Enzyme Specificity being found in the subfamilies GH13_1, 5, 6, 7, 15, 19, 24, 27, 28, 32, 36 and 37. The present in silico study delivers a proposal to create a novel GH13 subfamily with the Specificity of a-amylase. The proposal is based on a detailed bioinformatics analysis consisting of sequence, structural and evolutionary comparison of experimentally characterized a-amylases from, e.g., Bacillus aquimaris, Anoxybacillus sp. SK3-4 and DT3-1 and Geobacillus thermoleovorans , and hypothetical proteins, accompanied by a-amylases from well-established GH13 subfamilies and by closely related amylolytic Enzymes (mainly from the subfamily GH13_31). Three sequence-structural features can be ascribed to the members of the newly proposed GH13 subfamily: (i) the pair of adjacent tryptophan residues positioned between the CSR-V and CSR-II in the helix a3 of the catalytic TIM-barrel; (ii) the sequence LPDlx in their CSR-V; and (iii) a ~30-residue long C-terminal region with a motif of five conserved aromatic residues. From the evolutionary point of view, the novel GH13 a-amylase subfamily is most closely related to fungal and yeast a-amylases classified in the subfamily GH13_1.
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α-Amylase: an Enzyme Specificity found in various families of glycoside hydrolases
Cellular and Molecular Life Sciences, 2014Co-Authors: Stefan Janecek, Birte Svensson, E. Ann MacgregorAbstract:α-Amylase (EC 3.2.1.1) represents the best known amylolytic Enzyme. It catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch and related α-glucans. In general, the α-amylase is an Enzyme with a broad substrate preference and product Specificity. In the sequence-based classification system of all carbohydrate-active Enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α-amylase family, forms clan GH-H together with families GH70 and GH77 that, however, contain no α-amylase. Within the family GH13, the α-amylase Specificity is currently present in several subfamilies, such as GH13_1, 5, 6, 7, 15, 24, 27, 28, 36, 37, and, possibly in a few more that are not yet defined. The α-amylases classified in family GH13 employ a reaction mechanism giving retention of configuration, share 4–7 conserved sequence regions (CSRs) and catalytic machinery, and adopt the (β/α)_8-barrel catalytic domain. Although the family GH57 α-amylases also employ the retaining reaction mechanism, they possess their own five CSRs and catalytic machinery, and adopt a (β/α)_7-barrel fold. These family GH57 attributes are likely to be characteristic of α-amylases from the family GH119, too. With regard to family GH126, confirmation of the unambiguous presence of the α-amylase Specificity may need more biochemical investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases.
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Oligo-1,6-glucosidase and neopullulanase Enzyme subfamilies from the α-amylase family defined by the fifth conserved sequence region
Cellular and Molecular Life Sciences, 2002Co-Authors: A. Oslancová, Stefan JanecekAbstract:The α-amylase Enzyme family is the largest family of glycoside hydrolases. It contains almost 30 different Enzyme specificities covering hydrolases, transferases and isomerases. Some of the Enzyme specificities from the family are closely related, others less so. This study, based on the analysis of 79 amino acid sequences, postulates two subfamilies in the framework of the α-amylase family: the oligo-1,6-glucosidase subfamily and the neopullulanase subfamily. The specific sequence in the fifth conserved sequence region of the family served as the basis for defining the subfamilies: QpDln for the oligo-1,6-glucosidase subfamily and MPKln for the neopullulanase subfamily. This conserved sequence region is proposed to be the selection marker that enables one to distinguish between the two subfamilies. The 'intermediary' sequence MPDLN can be characteristic of the so-called intermediary group with a mixed Enzyme Specificity of α-amylase, cyclomaltodextrinase and neopullulanase. The evolutionary trees clearly supported the proposed definition of the two subfamilies.
Arren Bareven - One of the best experts on this subject based on the ideXlab platform.
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an in vivo metabolic approach for deciphering the product Specificity of glycerate kinase proves that both e coli s glycerate kinases generate 2 phosphoglycerate
PLOS ONE, 2015Co-Authors: Lior Zelcbuch, Manuel Razomejia, Elad Herz, Sagit Yahav, Niv Antonovsky, Hagar Kroytoro, Ron Milo, Arren BarevenAbstract:Apart from addressing humanity’s growing demand for fuels, pharmaceuticals, plastics and other value added chemicals, metabolic engineering of microbes can serve as a powerful tool to address questions concerning the characteristics of cellular metabolism. Along these lines, we developed an in vivo metabolic strategy that conclusively identifies the product Specificity of glycerate kinase. By deleting E. coli’s phosphoglycerate mutases, we divide its central metabolism into an ‘upper’ and ’lower’ metabolism, each requiring its own carbon source for the bacterium to grow. Glycerate can serve to replace the upper or lower carbon source depending on the product of glycerate kinase. Using this strategy we show that while glycerate kinase from Arabidopsis thaliana produces 3-phosphoglycerate, both E. coli’s Enzymes generate 2-phosphoglycerate. This strategy represents a general approach to decipher Enzyme Specificity under physiological conditions.
Tom Desmet - One of the best experts on this subject based on the ideXlab platform.
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identification of sucrose synthase in nonphotosynthetic bacteria and characterization of the recombinant Enzymes
Applied Microbiology and Biotechnology, 2015Co-Authors: Margo Diricks, Frederik De Bruyn, Paul Van Daele, Maarten Walmagh, Tom DesmetAbstract:Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into fructose and nucleotide (NDP)-glucose. To date, only SuSy’s from plants and cyanobacteria, both photosynthetic organisms, have been characterized. Here, four prokaryotic SuSy Enzymes from the nonphotosynthetic organisms Nitrosomonas Europaea (SuSyNe), Acidithiobacillus caldus (SuSyAc), Denitrovibrio acetiphilus (SusyDa), and Melioribacter roseus (SuSyMr) were recombinantly expressed in Escherichia coli and thoroughly characterized. The purified Enzymes were found to display high-temperature optima (up to 80 °C), high activities (up to 125 U/mg), and high thermostability (up to 15 min at 60 °C). Furthermore, SuSyAc, SuSyNe, and SuSyDa showed a clear preference for ADP as nucleotide, as opposed to plant SuSy’s which prefer UDP. A structural and mutational analysis was performed to elucidate the difference in NDP preference between eukaryotic and prokaryotic SuSy’s. Finally, the physiological relevance of this Enzyme Specificity is discussed in the context of metabolic pathways and genomic organization.
Manuel Razomejia - One of the best experts on this subject based on the ideXlab platform.
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an in vivo metabolic approach for deciphering the product Specificity of glycerate kinase proves that both e coli s glycerate kinases generate 2 phosphoglycerate
PLOS ONE, 2015Co-Authors: Lior Zelcbuch, Manuel Razomejia, Elad Herz, Sagit Yahav, Niv Antonovsky, Hagar Kroytoro, Ron Milo, Arren BarevenAbstract:Apart from addressing humanity’s growing demand for fuels, pharmaceuticals, plastics and other value added chemicals, metabolic engineering of microbes can serve as a powerful tool to address questions concerning the characteristics of cellular metabolism. Along these lines, we developed an in vivo metabolic strategy that conclusively identifies the product Specificity of glycerate kinase. By deleting E. coli’s phosphoglycerate mutases, we divide its central metabolism into an ‘upper’ and ’lower’ metabolism, each requiring its own carbon source for the bacterium to grow. Glycerate can serve to replace the upper or lower carbon source depending on the product of glycerate kinase. Using this strategy we show that while glycerate kinase from Arabidopsis thaliana produces 3-phosphoglycerate, both E. coli’s Enzymes generate 2-phosphoglycerate. This strategy represents a general approach to decipher Enzyme Specificity under physiological conditions.
Juliana Cardinalirezende - One of the best experts on this subject based on the ideXlab platform.
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the relevance of Enzyme Specificity for coEnzymes and the presence of 6 phosphogluconate dehydrogenase for polyhydroxyalkanoates production in the metabolism of pseudomonas sp lfm046
International Journal of Biological Macromolecules, 2020Co-Authors: Juliana Cardinalirezende, Alex Di Genova, Rafael A T P S Nahat, Alexander Steinbuchel, Mariefrance Sagot, Rafael S Costa, Henrique Da Costa Oliveira, Marilda Keico Taciro, Luiziana Ferreira Da SilvaAbstract:Reconstruction of genome-based metabolic model is a useful approach for the assessment of metabolic pathways, genes and proteins involved in the environmental fitness capabilities or pathogenic potential as well as for biotechnological processes development. Pseudomonas sp. LFM046 was selected as a good polyhydroxyalkanoates (PHA) producer from carbohydrates and plant oils. Its complete genome sequence and metabolic model were obtained. Analysis revealed that the gnd gene, encoding 6-phosphogluconate dehydrogenase, is absent in Pseudomonas sp. LFM046 genome. In order to improve the knowledge about LFM046 metabolism, the coEnzyme specificities of different Enzymes was evaluated. Furthermore, the heterologous expression of gnd genes from Pseudomonas putida KT2440 (NAD+ dependent) and Escherichia coli MG1655 (NADP+ dependent) in LFM046 was carried out and provoke a delay on cell growth and a reduction in PHA yield, respectively. The results indicate that the adjustment in cyclic Entner-Doudoroff pathway may be an interesting strategy for it and other bacteria to simultaneously meet divergent cell needs during cultivation phases of growth and PHA production.