Structural features underlying the selective cleavage of a novel exo-type maltose-forming amylase fromPyrococcussp. ST04

2014 ◽  
Vol 70 (6) ◽  
pp. 1659-1668 ◽  
Author(s):  
Kwang-Hyun Park ◽  
Jong-Hyun Jung ◽  
Sung-Goo Park ◽  
Myeong-Eun Lee ◽  
James F. Holden ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Basis ◽  
Amino Acid Sequence Identity ◽  
Structural Features ◽  
Industrial Applications ◽  
Closed Cavity ◽  
Helical Bundles ◽  
Sequence Identity ◽  
Hydrolysis Of ◽  
Potential Use

A novel maltose-forming α-amylase (PSMA) was recently found in the hyperthermophilic archaeonPyrococcussp. ST04. This enzyme shows <13% amino-acid sequence identity to other known α-amylases and displays a unique enzymatic property in that it hydrolyzes both α-1,4-glucosidic and α-1,6-glucosidic linkages of substrates, recognizing only maltose units, in an exo-type manner. Here, the crystal structure of PSMA at a resolution of 1.8 Å is reported, showing a tight ring-shaped tetramer with monomers composed of two domains: an N-domain (amino acids 1–341) with a typical GH57 family (β/α)7-barrel fold and a C-domain (amino acids 342–597) composed of α-helical bundles. A small closed cavity observed in proximity to the catalytic residues Glu153 and Asp253 at the domain interface has the appropriate volume and geometry to bind a maltose unit, accounting for the selective exo-type maltose hydrolysis of the enzyme. A narrow gate at the putative subsite +1 formed by residue Phe218 and Phe452 is essential for specific cleavage of glucosidic bonds. The closed cavity at the active site is connected to a short substrate-binding channel that extends to the central hole of the tetramer, exhibiting a geometry that is significantly different from classical maltogenic amylases or β-amylases. The structural features of this novel exo-type maltose-forming α-amylase provide a molecular basis for its unique enzymatic characteristics and for its potential use in industrial applications and protein engineering.

scholarly journals TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (8) ◽  
pp. 4266-4273 ◽  
Author(s):  
C H Ko ◽  
R F Gaber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid Sequence Identity ◽  
Functional Independence ◽  
Structural Features ◽  
Low Ph ◽  
Sequence Identity ◽  
Hydrophilic Region ◽  
Membrane Spanning ◽  
Delta Cells ◽  
Membrane Spanning Domains

We describe the cloning and molecular analysis of TRK2, the gene likely to encode the low-affinity K+ transporter in Saccharomyces cerevisiae. TRK2 encodes a protein of 889 amino acids containing 12 putative membrane-spanning domains (M1 through M12), with a large hydrophilic region between M3 and M4. These structural features closely resemble those contained in TRK1, the high-affinity K+ transporter. TRK2 shares 55% amino acid sequence identity with TRK1. The putative membrane-spanning domains of TRK1 and TRK2 share the highest sequence conservation, while the large hydrophilic regions between M3 and M4 exhibit the greatest divergence. The different affinities of TRK1 trk2 delta cells and trk1 delta TRK2 cells for K+ underscore the functional independence of the high- and low-affinity transporters. TRK2 is nonessential in TRK1 or trk1 delta haploid cells. The viability of cells containing null mutations in both TRK1 and TRK2 reveals the existence of an additional, functionally independent potassium transporter(s). Cells deleted for both TRK1 and TRK2 are hypersensitive to low pH; they are severely limited in their ability to take up K+, particularly when faced with a large inward-facing H+ gradient, indicating that the K+ transporter(s) that remains in trk1 delta trk2 delta cells functions differently than those of the TRK class.

scholarly journals Genetic and Biochemical Characterization of OXA-535, a Distantly Related OXA-48-Like β-Lactamase

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
L. Dabos ◽  
A. B. Jousset ◽  
R. A. Bonnin ◽  
N. Fortineau ◽  
A. Zavala ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequence Identity ◽  
Biochemical Characterization ◽  
Content Type ◽  
Sequence Identity

ABSTRACT OXA-535 is a chromosome-encoded carbapenemase of Shewanella bicestrii JAB-1 that shares only 91.3% amino acid sequence identity with OXA-48. Catalytic efficiencies are similar to those of OXA-48 for most β-lactams, except for ertapenem, where a 2,000-fold-higher efficiency was observed with OXA-535. OXA-535 and OXA-436, a plasmid-encoded variant of OXA-535 differing by three amino acids, form a novel cluster of distantly related OXA-48-like carbapenemases. Comparison of blaOXA-535 and blaOXA-436 genetic environments suggests that an ISCR1 may be responsible for blaOXA-436 gene mobilization from the chromosome of Shewanella spp. to plasmids.

TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (8) ◽  
pp. 4266-4273 ◽  
Author(s):  
C H Ko ◽  
R F Gaber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid Sequence Identity ◽  
Functional Independence ◽  
Structural Features ◽  
Low Ph ◽  
Sequence Identity ◽  
Hydrophilic Region ◽  
Membrane Spanning ◽  
Delta Cells ◽  
Membrane Spanning Domains

We describe the cloning and molecular analysis of TRK2, the gene likely to encode the low-affinity K+ transporter in Saccharomyces cerevisiae. TRK2 encodes a protein of 889 amino acids containing 12 putative membrane-spanning domains (M1 through M12), with a large hydrophilic region between M3 and M4. These structural features closely resemble those contained in TRK1, the high-affinity K+ transporter. TRK2 shares 55% amino acid sequence identity with TRK1. The putative membrane-spanning domains of TRK1 and TRK2 share the highest sequence conservation, while the large hydrophilic regions between M3 and M4 exhibit the greatest divergence. The different affinities of TRK1 trk2 delta cells and trk1 delta TRK2 cells for K+ underscore the functional independence of the high- and low-affinity transporters. TRK2 is nonessential in TRK1 or trk1 delta haploid cells. The viability of cells containing null mutations in both TRK1 and TRK2 reveals the existence of an additional, functionally independent potassium transporter(s). Cells deleted for both TRK1 and TRK2 are hypersensitive to low pH; they are severely limited in their ability to take up K+, particularly when faced with a large inward-facing H+ gradient, indicating that the K+ transporter(s) that remains in trk1 delta trk2 delta cells functions differently than those of the TRK class.

The yield of peptides and amino acids following acid hydrolysis of haemoglobin from porcine blood

2012 ◽  
Vol 52 (5) ◽  
pp. 313 ◽  
Author(s):  
Carlos Alvarez ◽  
Manuel Rendueles ◽  
Mario Diaz
Keyword(s):  
Amino Acids ◽  
Enzymatic Hydrolysis ◽  
Hydrochloric Acid ◽  
Waste Product ◽  
Good Method ◽  
Industrial Applications ◽  
Meat Industry ◽  
Experimental Conditions ◽  
Animal Blood ◽  
Hydrolysis Of

Animal blood is the most important waste product from the meat industry due to the huge volumes produced and its pollutant power. Different methods are currently employed to process this by-product, such as drying, incineration or enzymatic hydrolysis. All these techniques are expensive, do not result in revalorisation or are not applicable at an industrial scale. In this paper, chemical hydrolysis is presented as an alternative to recover and increase the value of purified haemoglobin, the most abundant protein in blood. Non-enzymatic hydrolysis of haemoglobin is a good method for obtaining peptides due to its low cost, ease of control and the large amount of peptides produced, as well as being suitable for industrial applications. This paper presents a study of the use of two acids (sulfuric and hydrochloric) for this purpose under different experimental conditions. From the analysis of the kinetics of the hydrolysis process, four fractions can be defined: unbroken haemoglobin, soluble peptides, non-soluble peptides and free amino acids. A kinetic model was developed to simulate the hydrolysis mechanisms, providing a good fit to the experimental results. Both sulfuric and hydrochloric acid at concentrations of 6 M can hydrolyse the haemoglobin completely, but the average peptide size is lower for sulfuric than for hydrochloric acid.

scholarly journals Structure of the family B DNA polymerase from the hyperthermophilic archaeonPyrobaculum calidifontis

2017 ◽  
Vol 73 (5) ◽  
pp. 420-427 ◽  
Author(s):  
Jingxu Guo ◽  
Wenling Zhang ◽  
Alun R. Coker ◽  
Steve P. Wood ◽  
Jonathan B. Cooper ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Polymerase ◽  
Active Sites ◽  
Dna Polymerases ◽  
Structural Features ◽  
Salt Bridges ◽  
Central Channel ◽  
High Sequence Identity ◽  
Sequence Identity ◽  
The Family

The family B DNA polymerase fromPyrobaculum calidifontis(Pc-polymerase) consists of 783 amino acids and is magnesium-ion dependent. It has an optimal pH of 8.5, an optimal temperature of 75°C and a half-life of 4.5 h at 95°C, giving it greater thermostability than the widely usedTaqDNA polymerase. The enzyme is also capable of PCR-amplifying larger DNA fragments of up to 7.5 kb in length. It was shown to have functional, error-correcting 3′–5′ exonuclease activity, as do the related high-fidelity DNA polymerases fromPyrococcus furiosus,Thermococcus kodakarensisKOD1 andThermococcus gorgonarius, which have extensive commercial applications.Pc-polymerase has a quite low sequence identity of approximately 37% to these enzymes, which, in contrast, have very high sequence identity to each other, suggesting that theP. calidifontisenzyme is distinct. Here, the structure determination ofPc-polymerase is reported, which has been refined to anRfactor of 24.47% and anRfreeof 28.81% at 2.80 Å resolution. The domains of the enzyme are arranged in a circular fashion to form a disc with a narrow central channel. One face of the disc has a number of connected crevices in it, which allow the protein to bind duplex and single-stranded DNA. The central channel is thought to allow incoming nucleoside triphosphates to access the active site. The enzyme has a number of unique structural features which distinguish it from other archaeal DNA polymerases and may account for its high processivity. A model of the complex with the primer-template duplex of DNA indicates that the largest conformational change that occurs upon DNA binding is the movement of the thumb domain, which rotates by 7.6° and moves by 10.0 Å. The surface potential of the enzyme is dominated by acidic groups in the central region of the molecule, where catalytic magnesium ions bind at the polymerase and exonuclease active sites. The outer regions are richer in basic amino acids that presumably interact with the sugar-phosphate backbone of DNA. The large number of salt bridges may contribute to the high thermal stability of this enzyme.

scholarly journals Cloning, Expression, and Characterization of a New β-Agarase fromVibriosp. Strain CN41

2011 ◽  
Vol 77 (19) ◽  
pp. 7077-7079 ◽  
Author(s):  
Li Liao ◽  
Xue-Wei Xu ◽  
Xia-Wei Jiang ◽  
Yi Cao ◽  
Na Yi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Glycoside Hydrolase ◽  
Amino Acid Sequence Identity ◽  
Sequence Identity

ABSTRACTA new agarase, AgaACN41, cloned fromVibriosp. strain CN41, consists of 990 amino acids, with only 49% amino acid sequence identity with known β-agarases. AgaACN41belongs to the GH50 (glycoside hydrolase 50) family but yields neoagarotetraose as the end product. AgaACN41was expressed and characterized.

scholarly journals Discovery of a Novel Glucagon-like Peptide (GCGL) and Its Receptor (GCGLR) in Chickens: Evidence for the Existence of GCGL and GCGLR Genes in Nonmammalian Vertebrates

Endocrinology ◽  
2012 ◽  
Vol 153 (11) ◽  
pp. 5247-5260 ◽  
Author(s):  
Yajun Wang ◽  
Fengyan Meng ◽  
Yu Zhong ◽  
Guian Huang ◽  
Juan Li
Keyword(s):  
Amino Acids ◽  
Spinal Cord ◽  
Amino Acid ◽  
Amino Acid Sequence Identity ◽  
Brain Regions ◽  
Rt Pcr ◽  
Adult Chicken ◽  
Sequence Identity ◽  
High Amino Acid ◽  
Glucagon Like Peptide

Abstract Glucagon (GCG), glucagon-related peptides, and their receptors have been reported to play important roles including the regulation of glucose homeostasis, gastrointestinal activity, and food intake in vertebrates. In this study, we identified genes encoding a novel glucagon-like peptide (named GCGL) and its receptor (GCGLR) from adult chicken brain using RACE and/or RT-PCR. GCGL was predicted to encode a peptide of 29 amino acids (cGCGL1–29), which shares high amino acid sequence identity with mammalian and chicken GCG (62–66%). GCGLR is a receptor of 430 amino acids and shares relatively high amino acid sequence identity (53–55%) with the vertebrate GCG receptor (GCGR). Using a pGL3-CRE-luciferase reporter system, we demonstrated that synthetic cGCGL1–29, but not its structurally related peptides, i.e. exendin-4 and GCG, could potently activate GCGLR (EC50: 0.10 nm) expressed in Chinese hamster ovary cells, indicating that GCGLR can function as a GCGL-specific receptor. RT-PCR assay revealed that GCGL expression is mainly restricted to several tissues including various brain regions, spinal cord, and testes, whereas GCGLR mRNA is widely expressed in adult chicken tissues with abundant expression noted in the pituitary, spinal cord, and various brain regions. Using synteny analysis, GCGL and GCGLR genes were also identified in the genomes of fugu, tetraodon, tilapia, medaka, coelacanth, and Xenopus tropicalis. As a whole, the discovery of GCGL and GCGLR genes in chickens and other nonmammalian vertebrates clearly indicates a previously unidentified role of GCGL-GCGLR in nonmammalian vertebrates and provides important clues to the evolutionary history of GCG and GCGL genes in vertebrates.

Deinking of recycled paper by coupling of the enzyme endo-β-1,4-D-glucanasa and the cellulose, and by using a laboratory flotation column

MRS Advances ◽  
2020 ◽  
Vol 5 (52-53) ◽  
pp. 2669-2678
Author(s):  
Jeovani González P. ◽  
Ramiro Escudero G
Keyword(s):  
Amino Acids ◽  
Sodium Hydroxide ◽  
Paper Industry ◽  
Computational Simulation ◽  
Cellulose Fibers ◽  
Residual Water ◽  
Chemical Reagent ◽  
Recycled Paper ◽  
Flotation Column ◽  
Hydrolysis Of

AbstractDeinking of recycled office (MOW) paper was carried out by using a flotation column and adding separately sodium hydroxide, and the enzyme Cellulase Thricodema Sp., as defibrillators.The de-inked cellulose fibers were characterized according to the standards of the paper industry, to compare the efficiency of the deinking of each chemical reagent used to hydrolyze the fibers and defibrillate them.The computational simulation of the molecular coupling between the enzyme and cellulose was performed, to establish the enzyme-cellulose molecular complex and then to identify the principal amino-acids of endo-β-1,4-D-glucanase in this molecular link, which are responsible for the hydrolysis of the cellulose.Experimental results show the feasibility to replace sodium hydroxide with the enzyme Cellulase Thricodema Sp., by obtaining deinked cellulose with similar optical and physical properties.The use of the enzyme instead of sodium hydroxide avoids the contamination of the residual water; in addition to that, the column is operated more easily, taking into consideration that the pH of the system goes from alkaline to neutral.

Studies on Chromatographic Fractioning by Cations Exchangers of a Bovine Hemoglobin Hydrolysate

2018 ◽  
Vol 69 (10) ◽  
pp. 2794-2798
Author(s):  
Alina Diana Panainte ◽  
Ionela Daniela Morariu ◽  
Nela Bibire ◽  
Madalina Vieriu ◽  
Gladiola Tantaru ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Retention Time ◽  
Chromatographic Separation ◽  
Bovine Hemoglobin ◽  
Reverse Phase ◽  
Hplc System ◽  
Fast Flow ◽  
Hydrolysis Of ◽  
Phase Column

A peptidic hydrolysate has been obtained through hydrolysis of bovine hemoglobin using pepsin. The fractioning of the hydrolysate was performed on a column packed with CM-Sepharose Fast Flow. The hydrolysate and each fraction was filtered and then injected into a HPLC system equipped with a Vydak C4 reverse phase column (0.46 x 25 cm), suitable for the chromatographic separation of large peptides with 20 to 30 amino acids. The detection was done using mass spectrometry, and the retention time, size and distribution of the peptides were determined.

scholarly journals The α-Chymotrypsin-catalyzed Hydrolysis of Esters of α-Amino Acids

1972 ◽  
Vol 247 (18) ◽  
pp. 5746-5752
Author(s):  
Ferenc J. Kézdy ◽  
Satya P. Jindal ◽  
Myron L. Bender
Keyword(s):  
Amino Acids ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of
Sign in / Sign up

Export Citation Format

Share Document