Enhancement of the Isomerization Activity and Thermostability of Cellobiose 2-Epimerase from Caldicellulosiruptor saccharolyticus by Exchange of a Flexible Loop

2021 ◽  
Vol 69 (6) ◽  
pp. 1907-1915
Author(s):  
Lu Wang ◽  
Jiali Gu ◽  
Yinghui Feng ◽  
Mingming Wang ◽  
Yanjun Tong ◽  
...  
Keyword(s):  
Caldicellulosiruptor Saccharolyticus ◽  
Flexible Loop

Insight into the potential factors influencing the catalytic direction in cellobiose 2-epimerase by crystallization and mutagenesis

2020 ◽  
Vol 76 (11) ◽  
pp. 1104-1113
Author(s):  
Yinghui Feng ◽  
Xiao Hua ◽  
Qiuyun Shen ◽  
Melissa Matthews ◽  
Yuzhu Zhang ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Structural Characteristics ◽  
Site Directed Mutagenesis ◽  
Caldicellulosiruptor Saccharolyticus ◽  
Flexible Loop ◽  
New Information ◽  
Hydrogen Bond Networks ◽  
Pharmaceutical Industries ◽  
Potential Factors

Cellobiose 2-epimerase (CE) is commonly recognized as an epimerase as most CEs mainly exhibit an epimerization activity towards disaccharides. In recent years, several CEs have been found to possess bifunctional epimerization and isomerization activities. They can convert lactose into lactulose, a high-value disaccharide that is widely used in the food and pharmaceutical industries. However, the factors that determine the catalytic direction in CEs are still not clear. In this study, the crystal structures of three newly discovered CEs, CsCE (a bifunctional CE from Caldicellulosiruptor saccharolyticus), StCE (a bifunctional CE from Spirochaeta thermophila DSM 6578) and BtCE (a monofunctional CE from Bacillus thermoamylovorans B4166), were determined at 1.54, 2.05 and 1.80 Å resolution, respectively, in order to search for structural clues to their monofunctional/bifunctional properties. A comparative analysis of the hydrogen-bond networks in the active pockets of diverse CEs, YihS and mannose isomerase suggested that the histidine corresponding to His188 in CsCE is uniquely required to catalyse isomerization. By alignment of the apo and ligand-bound structures of diverse CEs, it was found that bifunctional CEs tend to have more flexible loops and a larger entrance around the active site, and that the flexible loop 148–181 in CsCE displays obvious conformational changes during ligand binding. It was speculated that the reconstructed molecular interactions of the flexible loop during ligand binding helped to motivate the ligands to stretch in a manner beneficial for isomerization. Further site-directed mutagenesis analysis of the flexible loop in CsCE indicated that the residue composition of the flexible loop did not greatly impact epimerization but affects isomerization. In particular, V177D and I178D mutants showed a 50% and 80% increase in isomerization activity over the wild type. This study provides new information about the structural characteristics involved in the catalytic properties of CEs, which can be used to guide future molecular modifications.

Expression and Solution NMR Study of Multi-site Phosphomimetic Mutant BCL-2 Protein

2019 ◽  
Vol 26 (6) ◽  
pp. 449-457
Author(s):  
Ting Song ◽  
Keke Cao ◽  
Yu dan Fan ◽  
Zhichao Zhang ◽  
Zong W. Guo ◽  
...  
Keyword(s):  
Structural Change ◽  
Structural Biology ◽  
Quantum Coherence ◽  
Structural Changes ◽  
Solution Nmr ◽  
Flexible Loop ◽  
Loop Region ◽  
Loop Domain ◽  
Nmr Study ◽  
Binding Groove

Background: The significance of multi-site phosphorylation of BCL-2 protein in the flexible loop domain remains controversial, in part due to the lack of structural biology studies of phosphorylated BCL-2. Objective: The purpose of the study is to explore the phosphorylation induced structural changes of BCL-2 protein. Methods: We constructed a phosphomietic mutant BCL-2(62-206) (t69e, s70e and s87e) (EEEBCL- 2-EK (62-206)), in which the BH4 domain and the part of loop region was truncated (residues 2-61) to enable a backbone resonance assignment. The phosphorylation-induced structural change was visualized by overlapping a well dispersed 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy between EEE-BCL-2-EK (62-206) and BCL-2. Results: The EEE-BCL-2-EK (62-206) protein reproduced the biochemical and cellular activity of the native phosphorylated BCL-2 (pBCL-2), which was distinct from non-phosphorylated BCL-2 (npBCL-2) protein. Some residues in BH3 binding groove occurred chemical shift in the EEEBCL- 2-EK (62-206) spectrum, indicating that the phosphorylation in the loop region induces a structural change of active site. Conclusion: The phosphorylation of BCL-2 induced structural change in BH3 binding groove.

scholarly journals Structural Mechanism for Regulation of Bcl-2 protein Noxa by phosphorylation

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Christine B. Karim ◽  
L. Michel Espinoza-Fonseca ◽  
Zachary M. James ◽  
Eric A. Hanse ◽  
Jeffrey S. Gaynes ◽  
...  
Keyword(s):  
Md Simulations ◽  
Salt Bridges ◽  
Binding Assays ◽  
Peptide Backbone ◽  
Structural Mechanism ◽  
Binding Partner ◽  
Flexible Loop ◽  
Bh3 Domain ◽  
N Terminus ◽  
Β Sheets

Abstract We showed previously that phosphorylation of Noxa, a 54-residue Bcl-2 protein, at serine 13 (Ser13) inhibited its ability to promote apoptosis through interactions with canonical binding partner, Mcl-1. Using EPR spectroscopy, molecular dynamics (MD) simulations and binding assays, we offer evidence that a structural alteration caused by phosphorylation partially masks Noxa’s BH3 domain, inhibiting the Noxa-Mcl-1 interaction. EPR of unphosphorylated Noxa, with spin-labeled amino acid TOAC incorporated within the BH3 domain, revealed equilibrium between ordered and dynamically disordered states. Mcl-1 further restricted the ordered component for non-phosphorylated Noxa, but left the pSer13 Noxa profile unchanged. Microsecond MD simulations indicated that the BH3 domain of unphosphorylated Noxa is housed within a flexible loop connecting two antiparallel β-sheets, flanked by disordered N- and C-termini and Ser13 phosphorylation creates a network of salt-bridges that facilitate the interaction between the N-terminus and the BH3 domain. EPR showed that a spin label inserted near the N-terminus was weakly immobilized in unphosphorylated Noxa, consistent with a solvent-exposed helix/loop, but strongly constrained in pSer13 Noxa, indicating a more ordered peptide backbone, as predicted by MD simulations. Together these studies reveal a novel mechanism by which phosphorylation of a distal serine inhibits a pro-apoptotic BH3 domain and promotes cell survival.

scholarly journals Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway

2015 ◽  
Vol 290 (47) ◽  
pp. 28477-28488 ◽  
Author(s):  
Jana Kopečná ◽  
Israel Cabeza de Vaca ◽  
Nathan B. P. Adams ◽  
Paul A. Davison ◽  
Amanda A. Brindley ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Flexible Loop ◽  
Flow Through

scholarly journals Expression of Heterologous Cellulases inThermotogasp. Strain RQ2

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Hui Xu ◽  
Dongmei Han ◽  
Zhaohui Xu
Keyword(s):  
Enzyme Activities ◽  
Bacterial Cells ◽  
Recombinant Enzymes ◽  
Caldicellulosiruptor Saccharolyticus ◽  
Shuttle Vectors ◽  
E Coli ◽  
Chimeric Enzymes ◽  
Coding Regions ◽  
First Time ◽  
Culture Supernatants

The ability ofThermotogaspp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA) and Csac_1078 (celB) fromCaldicellulosiruptor saccharolyticuswere cloned intoT.sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA) and TM0070 (xynB), resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried byThermotoga-E. colishuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed inE. coliDH5αandT.sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. InE. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas inT.sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost inT.sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study) have ever been expressed inThermotoga, demonstrating the feasibility of using engineeredThermotogaspp. for efficient cellulose utilization.

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