scholarly journals Biochemical Characterization of the Amylase Activity from the New Haloarchaeal Strain Haloarcula sp. HS Isolated in the Odiel Marshlands

Biology ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 337
Author(s):  
Patricia Gómez-Villegas ◽  
Javier Vigara ◽  
Luis Romero ◽  
Cecilia Gotor ◽  
Sara Raposo ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Amylase Activity ◽  
Extreme Environments ◽  
Three Dimensional ◽  
Large Family ◽  
Industrial Applications ◽  
Dimensional Structure ◽  
Glycosyl Hydrolases ◽  
Calcium Dependent ◽  
Alpha Amylases

Alpha-amylases are a large family of α,1-4-endo-glycosyl hydrolases distributed in all kingdoms of life. The need for poly-extremotolerant amylases encouraged their search in extreme environments, where archaea become ideal candidates to provide new enzymes that are able to work in the harsh conditions demanded in many industrial applications. In this study, a collection of haloarchaea isolated from Odiel saltern ponds in the southwest of Spain was screened for their amylase activity. The strain that exhibited the highest activity was selected and identified as Haloarcula sp. HS. We demonstrated the existence in both, cellular and extracellular extracts of the new strain, of functional α-amylase activities, which showed to be moderately thermotolerant (optimum around 60 °C), extremely halotolerant (optimum over 25% NaCl), and calcium-dependent. The tryptic digestion followed by HPLC-MS/MS analysis of the partially purified cellular and extracellular extracts allowed to identify the sequence of three alpha-amylases, which despite sharing a low sequence identity, exhibited high three-dimensional structure homology, conserving the typical domains and most of the key consensus residues of α-amylases. Moreover, we proved the potential of the extracellular α-amylase from Haloarcula sp. HS to treat bakery wastes under high salinity conditions.

scholarly journals Protein Phosphatase 2B (PP2B, Calcineurin) in Paramecium: Partial Characterization Reveals That Two Members of the Unusually Large Catalytic Subunit Family Have Distinct Roles in Calcium-Dependent Processes

2010 ◽  
Vol 9 (7) ◽  
pp. 1049-1063 ◽  
Author(s):  
D. Fraga ◽  
I. M. Sehring ◽  
R. Kissmehl ◽  
M. Reiss ◽  
R. Gaines ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Amino Acid Level ◽  
Three Dimensional ◽  
Large Family ◽  
Genome Project ◽  
Dimensional Structure ◽  
Sequence Information ◽  
Paramecium Tetraurelia ◽  
Content Type ◽  
Calcium Dependent

ABSTRACT We characterized the calcineurin (CaN) gene family, including the subunits CaNA and CaNB, based upon sequence information obtained from the Paramecium genome project. Paramecium tetraurelia has seven subfamilies of the catalytic CaNA subunit and one subfamily of the regulatory CaNB subunit, with each subfamily having two members of considerable identity on the amino acid level (≥55% between subfamilies, ≥94% within CaNA subfamilies, and full identity in the CaNB subfamily). Within CaNA subfamily members, the catalytic domain and the CaNB binding region are highly conserved and molecular modeling revealed a three-dimensional structure almost identical to a human ortholog. At 14 members, the size of the CaNA family is unprecedented, and we hypothesized that the different CaNA subfamily members were not strictly redundant and that at least some fulfill different roles in the cell. This was tested by selecting two phylogenetically distinct members of this large family for posttranscriptional silencing by RNA interference. The two targets resulted in differing effects in exocytosis, calcium dynamics, and backward swimming behavior that supported our hypothesis that the large, highly conserved CaNA family members are not strictly redundant and that at least two members have evolved diverse but overlapping functions. In sum, the occurrence of CaN in Paramecium spp., although disputed in the past, has been established on a molecular level. Its role in exocytosis and ciliary beat regulation in a protozoan, as well as in more complex organisms, suggests that these roles for CaN were acquired early in the evolution of this protein family.

scholarly journals Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins

1993 ◽  
Vol 2 (3) ◽  
pp. 366-382 ◽  
Author(s):  
Miroslaw Cygler ◽  
Joseph D. Schrag ◽  
Joel L. Sussman ◽  
Michal Harel ◽  
Israel Silman ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Large Family ◽  
Sequence Conservation ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Related Proteins

scholarly journals A computer-based approach for developing linamarase inhibitory agents

2020 ◽  
Vol 5 (7) ◽  
Author(s):  
Lucas Paul ◽  
Celestin N. Mudogo ◽  
Kelvin M. Mtei ◽  
Revocatus L. Machunda ◽  
Fidele Ntie-Kang
Keyword(s):  
Structure Elucidation ◽  
Three Dimensional ◽  
Data Bank ◽  
Competitive Inhibitor ◽  
Industrial Applications ◽  
Dimensional Structure ◽  
Full Understanding ◽  
The Poor ◽  
Computer Based ◽  
Inhibitory Agents

AbstractCassava is a strategic crop, especially for developing countries. However, the presence of cyanogenic compounds in cassava products limits the proper nutrients utilization. Due to the poor availability of structure discovery and elucidation in the Protein Data Bank is limiting the full understanding of the enzyme, how to inhibit it and applications in different fields. There is a need to solve the three-dimensional structure (3-D) of linamarase from cassava. The structural elucidation will allow the development of a competitive inhibitor and various industrial applications of the enzyme. The goal of this review is to summarize and present the available 3-D modeling structure of linamarase enzyme using different computational strategies. This approach could help in determining the structure of linamarase and later guide the structure elucidation in silico and experimentally.

scholarly journals DUF3380 Domain from a Salmonella Phage Endolysin Shows PotentN-Acetylmuramidase Activity

2016 ◽  
Vol 82 (16) ◽  
pp. 4975-4981 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Hans Gerstmans ◽  
Simon Thorpe ◽  
Stéphane Mesnage ◽  
Rob Lavigne ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Thermal Resistance ◽  
High Performance ◽  
Antimicrobial Agents ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Gram Negative ◽  
Content Type

ABSTRACTBacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novelSalmonellabacteriophage endolysin, Gp110, which comprises an uncharacterizeddomain ofunknownfunction (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 hasN-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond betweenN-acetylmuramic acid andN-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents.IMPORTANCEWe report the functional and biochemical characterization of theSalmonellaphage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 hasN-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

Structure of the Calcium Pump from Sarcoplasmic Reticulum at 8 Å Resolution: Architecture of the Transmembrane Helices and Localization of the Binding Site for Thapsigargin

1998 ◽  
Vol 4 (S2) ◽  
pp. 462-463
Author(s):  
P. Zhang ◽  
C. Toyoshima ◽  
K. Yonekura ◽  
G. Inesi ◽  
M. Green ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Three Dimensional ◽  
Specific Inhibitor ◽  
Large Family ◽  
Calcium Pump ◽  
Dimensional Structure ◽  
Transmembrane Helices ◽  
Site Mutagenesis ◽  
P Type

The calcium pump (Ca2+-ATPase) from sarcoplasmic reticulum (SR) is a prominent member of the large family of ATP-dependent cation pumps, which include Na+ /K+-ATPase, H+/K+-ATPase from the stomach, H+-ATPase from yeast and Neurospora, and various detoxifying pumps for Cd+, Cu+ and other metals. In muscle, calcium is stored inside the SR and contraction is initiated by regulated release through specific calcium channels; Ca2+ -ATPase is responsible for relaxation by pumping calcium back into the SR lumen. Many techniques (chemical modification, site mutagenesis, reaction kinetics) have been used to correlate Ca2+-ATPase sequence with function, but no high resolution three-dimensional structure of Ca2+-ATPase, or any P-type pump, has yet been determined. In the current work, we have determined the structure from helical crystals at 8 A resolution and thus revealed the alpha-helical architecture of the transmembrane domain. In addition, a specific inhibitor of Ca2+-ATPase, thapsigargin, was used to promote crystallization and we have characterized the structural consequences of its inhibition.

scholarly journals Induction of Rare Conformation of Oligosaccharide by Binding to Calcium-dependent Bacterial Lectin: X-ray Crystallography and Modelling Study

2019 ◽  
Author(s):  
Martin Lepsik ◽  
Roman Sommer ◽  
Sakonwan Kuhaudomlarp ◽  
Mickaёl Lelimousin ◽  
Emanuele Paci ◽  
...  
Keyword(s):  
Force Field ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Lewis X ◽  
X Ray ◽  
X Ray Crystallography ◽  
Calcium Dependent ◽  
Protein Receptors ◽  
Dependent Protein ◽  
Micro Organisms

ABSTRACTPathogenic micro-organisms utilize protein receptors in adhesion to host tissues, a process that in some cases relies on the interaction between lectin and human glycoconjugates. Oligosaccharide epitopes are recognized through their three-dimensional structure and their flexibility is a key issue in specificity. In this paper, we analyse by X-ray crystallography the structures of the lectin LecB from two strains of Pseudomonas aeruginosa in complex with Lewis x oligosaccharide present on cell surfaces of human tissues. An unusual conformation of the glycan was observed in all binding sites with a non-canonical syn orientation of the N-acetyl group of N-acetyl-glucosamine. A PDB-wide search revealed that such an orientation occurs only in 2% of protein/carbohydrate complexes. Theoretical chemistry calculations showed that the observed conformation is unstable in solution but stabilised by the lectin. A reliable description of LecB/Lewis x complex by force field-based methods had proven as especially challenging due to the special feature of the binding site, two closely apposed Ca2+ ions which induce strong charge delocalisation. By comparing various force-field parametrisations, we design general protocols which will be useful in near future for designing carbohydrate-based ligands (glycodrugs) against other calcium-dependent protein receptors.

scholarly journals Structure and Functional Analysis of a Ribosomal Oxygenase

2014 ◽  
Vol 70 (a1) ◽  
pp. C1408-C1408
Author(s):  
Laura van Staalduinen ◽  
Stefanie Novakowski ◽  
Zongchao Jia
Keyword(s):  
Ribosomal Protein ◽  
Active Site ◽  
Three Dimensional ◽  
Large Family ◽  
Structural Similarity ◽  
Dimensional Structure ◽  
Titration Calorimetry ◽  
Ribosome Assembly ◽  
Oxidation Reactions ◽  
Jmjc Domain

The 2-oxoglutarate/Fe(II)-dependent oxygenases (2OG oxygenases) are a large family of proteins that share a similar overall three-dimensional structure and catalyze a diverse array of oxidation reactions. The Jumonji C (JmjC)-domain containing proteins represent an important subclass of the 2OG oxygenase family that typically catalyze protein hydroxylation; however, recently other reactions have been identified, such as tRNA modification. The E. coli gene, ycfD, was predicted to be a JmjC-domain containing protein of unknown function based on primary sequence. Recently YcfD was determined to act as a ribosomal oxygenase, hydroxylating an arginine residue on the 50S ribosomal protein L-16 (RL-16). We have determined the crystal structure of YcfD at 2.7 Å resolution, revealing that YcfD is structurally similar to known JmjC proteins and possesses the characteristic double stranded β-helix fold or cupin domain. Separate from the cupin domain, an additional globular module termed -helical arm mediates dimerization of YcfD. We further have shown that 2-oxoglutarate binds to YcfD using isothermal titration calorimetry and identified R140 and S116 as key 2OG binding residues using mutagenesis which, together with the iron location and structural similarity with other cupin family members, allowed identification of the active site. Structural homology to ribosomal assembly proteins combined with GST-YcfD pull-down of a ribosomal protein and docking of RL-16 to the YcfD active site support the role of YcfD in regulation of bacterial ribosome assembly. Furthermore, overexpression of YcfD is shown to inhibit cell growth signifying a toxic effect on ribosome assembly.

scholarly journals High-resolution crystal structure and biochemical characterization of a GH11 endoxylanase from Nectria haematococca

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hina Andaleeb ◽  
Najeeb Ullah ◽  
Sven Falke ◽  
Markus Perbandt ◽  
Hévila Brognaro ◽  
...  
Keyword(s):  
Alternative Fuels ◽  
Biochemical Characterization ◽  
Three Dimensional ◽  
Detailed Comparison ◽  
Structural Features ◽  
Dimensional Structure ◽  
Nectria Haematococca ◽  
Chemical Reagents ◽  
Beechwood Xylan

Abstract Enzymatic degradation of vegetal biomass offers versatile procedures to improve the production of alternative fuels and other biomass-based products. Here we present the three-dimensional structure of a xylanase from Nectria haematococca (NhGH11) at 1.0 Å resolution and its functional properties. The atomic resolution structure provides details and insights about the complex hydrogen bonding network of the active site region and allowed a detailed comparison with homologous structures. Complementary biochemical studies showed that the xylanase can catalyze the hydrolysis of complex xylan into simple xylose aldopentose subunits of different lengths. NhGH11 can catalyze the efficient breakdown of beechwood xylan, xylan polysaccharide, and wheat arabinoxylan with turnover numbers of 1730.6 ± 318.1 min−1, 1648.2 ± 249.3 min−1 and 2410.8 ± 517.5 min−1 respectively. NhGH11 showed maximum catalytic activity at pH 6.0 and 45 °C. The mesophilic character of NhGH11 can be explained by distinct structural features in comparison to thermophilic GH11 enzymes, including the number of hydrogen bonds, side chain interactions and number of buried water molecules. The enzymatic activity of NhGH11 is not very sensitive to metal ions and chemical reagents that are typically present in associated industrial production processes. The data we present highlights the potential of NhGH11 to be applied in industrial biomass degradation processes.

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