Structure and function of the type III pullulan hydrolase fromThermococcus kodakarensis

2018 ◽  
Vol 74 (4) ◽  
pp. 305-314 ◽  
Author(s):  
Jingxu Guo ◽  
Alun R. Coker ◽  
Steve P. Wood ◽  
Jonathan B. Cooper ◽  
Ronan M. Keegan ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Catalytic Domain ◽  
Disulfide Bridge ◽  
Salt Bridges ◽  
Calcium Binding Site ◽  
Ph Optimum ◽  
Type Iii ◽  
Terminal Domain ◽  
Industry Type ◽  
And Function

Pullulan-hydrolysing enzymes, more commonly known as debranching enzymes for starch and other polysaccharides, are of great interest and have been widely used in the starch-saccharification industry. Type III pullulan hydrolase fromThermococcus kodakarensis(TK-PUL) possesses both pullulanase and α-amylase activities. Until now, only two enzymes in this class, which are capable of hydrolysing both α-1,4- and α-1,6-glycosidic bonds in pullulan to produce a mixture of maltose, panose and maltotriose, have been described. TK-PUL shows highest activity in the temperature range 95–100°C and has a pH optimum in the range 3.5–4.2. Its unique ability to hydrolyse maltotriose into maltose and glucose has not been reported for other homologous enzymes. The crystal structure of TK-PUL has been determined at a resolution of 2.8 Å and represents the first analysis of a type III pullulan hydrolyse. The structure reveals that the last part of the N-terminal domain and the C-terminal domain are significantly different from homologous structures. In addition, the loop regions at the active-site end of the central catalytic domain are quite different. The enzyme has a well defined calcium-binding site and possesses a rare vicinal disulfide bridge. The thermostability of TK-PUL and its homologues may be attributable to several factors, including the increased content of salt bridges, helical segments, Pro, Arg and Tyr residues and the decreased content of serine.

scholarly journals A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate

FEBS Open Bio ◽  
2016 ◽  
Vol 6 (5) ◽  
pp. 425-432 ◽  
Author(s):  
Johannes Then ◽  
Ren Wei ◽  
Thorsten Oeser ◽  
André Gerdts ◽  
Juliane Schmidt ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Binding Site ◽  
Polyethylene Terephthalate ◽  
Calcium Binding ◽  
Disulfide Bridge ◽  
Calcium Binding Site

scholarly journals Functional characterization of the non-catalytic ectodomains of the nucleotide pyrophosphatase/phosphodiesterase NPP1

2003 ◽  
Vol 371 (2) ◽  
pp. 321-330 ◽  
Author(s):  
Rik GIJSBERS ◽  
Hugo CEULEMANS ◽  
Mathieu BOLLEN
Keyword(s):  
Catalytic Activity ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Functional Characterization ◽  
Structure Stability ◽  
Subunit Structure ◽  
Terminal Domain ◽  
Nucleotide Pyrophosphatase ◽  
Domain Truncation ◽  
And Function

The ubiquitous nucleotide pyrophosphatases/phosphodiesterases NPP1–3 consist of a short intracellular N-terminal domain, a single transmembrane domain and a large extracellular part, comprising two somatomedin-B-like domains, a catalytic domain and a poorly defined C-terminal domain. We show here that the C-terminal domain of NPP1–3 is structurally related to a family of DNA/RNA non-specific endonucleases. However, none of the residues that are essential for catalysis by the endonucleases are conserved in NPP1–NPP3, suggesting that the nuclease-like domain of NPP1–3 does not represent a second catalytic domain. Truncation analysis revealed that the nuclease-like domain of NPP1 is required for protein stability, for the targeting of NPP1 to the plasma membrane and for the expression of catalytic activity. We also demonstrate that 16 conserved cysteines in the somatomedin-B-like domains of NPP1, in concert with two flanking cysteines, mediate the dimerization of NPP1. The K173Q polymorphism of NPP1, which maps to the second somatomedin-B-like domain and has been associated with the aetiology of insulin resistance, did not affect the dimerization or catalytic activity of NPP1, and did not endow NPP1 with an affinity for the insulin receptor. Our data suggest that the non-catalytic ectodomains contribute to the subunit structure, stability and function of NPP1–3.

TMEM16A protein: calcium binding site and its activation mechanism

2021 ◽  
Vol 28 ◽  
Author(s):  
Wanying Ji ◽  
Donghong Shi ◽  
Sai Shi ◽  
Xiao Yang ◽  
Yafei Chen ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Calcium Ions ◽  
Calcium Binding ◽  
Molecular Design ◽  
Chloride Ion ◽  
Activation Mechanism ◽  
Calcium Binding Site ◽  
Key Issues ◽  
Function Relationship ◽  
And Function

: TMEM16A mediates calcium-activated transmembrane flow of chloride ion and a variety of physiological functions. The binding of cytoplasmic calcium ions of TMEM16A and the consequent conformational changes of it are the key issues to explore the relationship between its structure and function. In recent years, researchers have explored this issue through electrophysiological experiment, structure resolving, molecular dynamic simulation and other methods. The structures of TMEM16 family members resolved by cryo-Electron microscopy (cryo-EM) and X-ray crystallization provide the primarily basis for the investigation of the molecular mechanism of TMEM16A. However, the binding and activation mechanism of calcium ions in TMEM16A are still unclear and controversial. This review discusses four Ca2+ sensing sites of TMEM16A and analyze activation properties of TMEM16A by them, which will help to understand the structure-function relationship of TMEM16A and throw light on the molecular design targeting TMEM16A channel.

Studies On Calcium-Activated Neutral Protease In Platelet

1981 ◽  
Author(s):  
M Sakon ◽  
H Ohno ◽  
E Kurokawa ◽  
J Kambayashi ◽  
G Kōsaki
Keyword(s):  
Calcium Binding ◽  
Soluble Fraction ◽  
Active Form ◽  
Physiological Role ◽  
Neutral Protease ◽  
Calcium Binding Site ◽  
Ph Optimum ◽  
Inactive Form ◽  
Caseinolytic Activity ◽  
Platelet Reaction

It has been reported that platelet aggregation or release of arachidonic. acid in stimulated platelets is inhibited by protease inhibitors or by synthetic substrates for proteases, suggesting possible involvement of unknown proteases in the platelet reaction. The present study was initiated to identify such proteases in platelets.Subcellular fractions of bovine platelets were obtained according to the method of Barber and Jamieson and caseinolytic activity was studied in each fraction at pH 8.0 in the presence or absence of Ca2+, using fluorescamine method. A significant caseinolytic activity was detected in the soluble fraction but none in the fractions of plasma membrane or pellet. Then, this protease was partially purified from the soluble fraction by means of ammonium sulfate fractionation and subsequent column chromatographies on DEAE-Sepharose CL-6B and Sephadex G-150. The partially purified protease required Ca2+ strictly for its activity with the optimal concentration of 1 mM and pH optimum for the activity was 8.0, by which this enzyme was classified as a calcium-activated neutral protease. The molecular weight was estimated to be 135,000 by Andrew’s method. The activity of the protease was strongly inhibited by sulfhydryl reagents but no enhancement of the activity was observed in the presence of cysteine or reduced glutathione. The inhibition of the activity by PMSF or DFP was observed only when the protease was preincubated at 25°C for 1 hour in the presence of Ca2+.From these observations, it may be suggested that the enzyme may possess calcium binding site independent of the catalytic site and that by changing the conformation of the enzyme Ca2+ may convert the enzyme from an inactive form to an active form. However, the physiological role of the enzyme in the platelet reaction has yet to be fully elucidated.

scholarly journals X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure–function studies of a neuronal calcium-signalling protein

2015 ◽  
Vol 71 (3) ◽  
pp. 615-631 ◽  
Author(s):  
P. T. Erskine ◽  
A. Fokas ◽  
C. Muriithi ◽  
H. Rehman ◽  
L. A. Yates ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Normal Mode ◽  
Calcium Ions ◽  
Calcium Binding ◽  
Calcium Binding Site ◽  
X Ray ◽  
Terminal Domain ◽  
Ef Hand ◽  
Ef Hands ◽  
Photoreceptor Neurons

The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squidLoligo pealeishowed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly,L. pealeicalexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the protein has two markedly different affinities for calcium ions, the weaker of which is most likely to be associated with binding of calcium ions to the protein during neuronal excitation. In the current study, site-directed mutagenesis has been used to abolish each of the three calcium-binding sites of calexcitin, and these experiments suggest that it is the single calcium-binding site in the C-terminal domain of the protein which is likely to have a sensory role in the neuron.

Endoglucanase G from Fibrobacter succinogenes S85 belongs to a class of enzymes characterized by a basic C-terminal domain

1996 ◽  
Vol 42 (9) ◽  
pp. 934-943 ◽  
Author(s):  
Abiye H. Iyo ◽  
Cecil W. Forsberg
Keyword(s):  
Catalytic Domain ◽  
Specific Activity ◽  
Glutamate Synthase ◽  
Open Reading Frames ◽  
Crystalline Cellulose ◽  
Fibrobacter Succinogenes ◽  
Ph Optimum ◽  
E Coli ◽  
Terminal Domain ◽  
Orf2 Protein

A 3.6-kb fragment of the Fibrobacter succinogenes S85 DNA was sequenced and found to contain two open reading frames (ORFs) on the same strand separated by 242 nucleotide bases. The translated protein from ORF1 had a predicted mass of 52.3 kDa. In a region of 320 amino acid overlap, it shares a 35% identity with the b-chain of the glutamate synthase of Escherichia coli. The ORF2 protein encodes a 519 residue protein designated CelG. It consists of an ORF of 1557 bp, encoding a polypeptide of 54.5 kDa. The N-terminal region, which contains the catalytic domain, is linked to a C-terminal basic domain, which has a predicted isoelectric point of 10.8. The catalytic domain in endoglucanase G (CelG) is homologous to the family 5 (A) cellulases. The enzyme has an apparent mass of 55 kDa, a pH optimum of 5.5, and temperature optimum of 25 °C. It had a specific activity of 16.5 mmol∙min−1∙mg−1 on barley b-glucan and produced a mixture of cellooligosaccharides from the hydrolysis of acid swollen cellulose and cellooligosaccharides. Antiserum raised against the purified form of CelG in E. coli failed to react with proteins from the native organism when grown on either glucose or crystalline cellulose, but reverse transcription and polymerase chain reaction techniques using RNA from the native organism demonstrated that the celG gene was expressed constitutively. Its distribution amongst subspecies of Fibrobacter was restricted to F. succinogenes S85.Key words: basic terminal domain, Fibrobacter succinogenes, endoglucanase, nucleotide sequence.

scholarly journals A topologically distinct class of photolyases specific for UV lesions within single-stranded DNA

2020 ◽  
Vol 48 (22) ◽  
pp. 12845-12857
Author(s):  
Hans-Joachim Emmerich ◽  
Martin Saft ◽  
Leonie Schneider ◽  
Dennis Kock ◽  
Alfred Batschauer ◽  
...  
Keyword(s):  
Flavin Adenine Dinucleotide ◽  
Catalytic Domain ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Single Stranded Dna ◽  
Dna Damages ◽  
Terminal Domain ◽  
And Function ◽  
Uv Lesions ◽  
Fad Binding

Abstract Photolyases are ubiquitously occurring flavoproteins for catalyzing photo repair of UV-induced DNA damages. All photolyases described so far have a bilobal architecture with a C-terminal domain comprising flavin adenine dinucleotide (FAD) as catalytic cofactor and an N-terminal domain capable of harboring an additional antenna chromophore. Using sequence-similarity network analysis we discovered a novel subgroup of the photolyase/cryptochrome superfamily (PCSf), the NewPHLs. NewPHL occur in bacteria and have an inverted topology with an N-terminal catalytic domain and a C-terminal domain for sealing the FAD binding site from solvent access. By characterizing two NewPHL we show a photochemistry characteristic of other PCSf members as well as light-dependent repair of CPD lesions. Given their common specificity towards single-stranded DNA many bacterial species use NewPHL as a substitute for DASH-type photolyases. Given their simplified architecture and function we suggest that NewPHL are close to the evolutionary origin of the PCSf.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

Structure prediction of SPAK C-terminal domain and analysis of its binding to RFXV/I motifs by homology modelling, docking, and molecular dynamics simulation studies

2020 ◽  
Vol 16 ◽  
Author(s):  
Mubarak A. Alamri ◽  
Ahmed D. Alafnan ◽  
Obaid Afzal ◽  
Alhumaidi B. Alabbas ◽  
Safar M. Alqahtani
Keyword(s):  
Molecular Dynamic ◽  
Dynamic Stability ◽  
Md Simulation ◽  
Homology Modelling ◽  
Antihypertensive Agents ◽  
Structure And Function ◽  
Dynamics Simulation ◽  
Dimensional Structure ◽  
Terminal Domain ◽  
And Function

Background: The STE20/SPS1-related proline/alanine-rich kinase (SPAK) is a component of WNKSPAK/OSR1 signaling pathway that plays an essential role in blood pressure regulation. The function of SPAK is mediated by its highly conserved C-terminal domain (CTD) that interacts with RFXV/I motifs of upstream activators, WNK kinases, and downstream substrate, cation-chloride cotransporters. Objective: To determine and validate the three-dimensional structure of the CTD of SPAK and to study and analyze its interaction with the RFXV/I motifs. Methods: A homology model of SPAK CTD was generated and validated through multiple approaches. The model was based on utilizing the OSR1 protein kinase as a template. This model was subjected to 100 ns molecular dynamic (MD) simulation to evaluate its dynamic stability. The final equilibrated model was used to dock the RFQV-peptide derived from WNK4 into the primary pocket that was determined based on the homology sequence between human SPAK and OSR1 CTDs. The mechanism of interaction, conformational rearrangement and dynamic stability of the binding of RFQV-peptide to SPAK CTD were characterized by molecular docking and molecular dynamic simulation. Results: The MD simulation suggested that the binding of RFQV induces a large conformational change due to the distribution of salt bridge within the loop regions. These results may help in understanding the relation between the structure and function of SPAK CTD and to support drug design of potential SPAK kinase inhibitors as antihypertensive agents. Conclusion: This study provides deep insight into SPAK CTD structure and function relationship.

Sign in / Sign up

Export Citation Format

Share Document