scholarly journals Role of E270 in pH- and metal-sensitivities of firefly luciferases

2020 ◽  
Vol 19 (11) ◽  
pp. 1548-1558
Author(s):  
V. R. Viviani ◽  
G. F. Pelentir ◽  
G. Oliveira ◽  
A. Tomazini ◽  
V. R. Bevilaqua
Keyword(s):  
Active Site ◽  
Ph Sensitive ◽  
Ph Sensitivity ◽  
Decreasing Ph ◽  
Α Helix ◽  
Conserved Residue ◽  
Active Site Conformation

The substitutions of the conserved residue E270 at the N-terminal of α-helix 10 in pH-sensitive firefly luciferases stabilize a green emitting active site conformation, decreasing pH-sensitivity.

scholarly journals Probing the flavin transfer mechanism in alkanesulfonate monooxygenase system

2018 ◽  
Author(s):  
PV Dayal ◽  
HR Ellis
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Lag Phase ◽  
Monooxygenase System ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Loop Region ◽  
Alkanesulfonate Monooxygenase ◽  
Α Helix

AbstractBacteria acquire sulfur through the sulfur assimilation pathway, but under sulfur limiting conditions bacteria must acquire sulfur from alternative sources. The alkanesulfonate monooxygenase enzymes are expressed under sulfur-limiting conditions, and catalyze the desulfonation of wide-range of alkanesulfonate substrates. The SsuE enzyme is an NADPH-dependent FMN reductase that provides reduced flavin to the SsuD monooxygenase. The mechanism for the transfer of reduced flavin in flavin dependent two-component systems occurs either by free-diffusion or channeling. Previous studies have shown the presence of protein-protein interactions between SsuE and SsuD, but the identification of putative interaction sights have not been investigated. Current studies utilized HDX-MS to identify protective sites on SsuE and SsuD. A conserved α-helix on SsuD showed a decrease in percent deuteration when SsuE was included in the reaction. This suggests the role of α-helix in promoting protein-protein interactions. Specific SsuD variants were generated in order to investigate the role of these residues in protein-protein interactions and catalysis. Variant containing substitutions at the charged residues showed a six-fold decrease in the activity, while a deletion variant of SsuD lacking the α-helix showed no activity when compared to wild-type SsuD. In addition, there was no protein-protein interactions identified between SsuE and his-tagged SsuD variants in pull-down assays, which correlated with an increase in the Kd value. The α-helix is located right next to a dynamic loop region, positioned at the entrance of the active site. The putative interaction site and dynamic loop region located so close to the active site of SsuD suggests the importance of this region in the SsuD catalysis. Stopped-flow studies were performed to analyze the lag-phase which signifies the stabilization and transfer of reduced flavin from SsuE to SsuD. The SsuD variants showed a decrease in lag-phase, which could be because of a downturn in flavin transfer. A competitive assay was devised to evaluate the mechanism of flavin transfer in the alkanesulfonate monooxygenase system. A variant of SsuE was generated which interacted with SsuD, but was not able to reduce FMN. Assays that included varying concentrations of Y118A SsuE and wild-type SsuE in the coupled assays showed a decrease in the desulfonation activity of SsuD. The decrease in activity could be by virtue of Y118A SsuE competing with the wild-type SsuE for the putative docking site on SsuD. These studies define the importance of protein-protein interactions for the efficient transfer of reduced flavin from SsuE to SsuD leading to the desulfonation of alkanesulfonates.

Heparan sulphate N-sulphotransferase activity: reaction mechanism and substrate recognition

2003 ◽  
Vol 31 (2) ◽  
pp. 331-334 ◽  
Author(s):  
Y. Kakuta ◽  
L. Li ◽  
L.C. Pedersen ◽  
L.G. Pedersen ◽  
M. Negishi
Keyword(s):  
Active Site ◽  
Transfer Reaction ◽  
Substrate Recognition ◽  
Heparan Sulphate ◽  
Critical Role ◽  
Random Coil ◽  
Group Transfer ◽  
Α Helix ◽  
Model Chain

Human heparan sulphate N-deacetylase/N-sulphotransferase 1 sulphates the NH3+ group of the glucosamine moiety of the heparan chain in heparan sulphate/heparin biosynthesis. An open cleft that runs perpendicular to the sulphate donor 3´-phosphoadenosine 5´-phosphosulphate may constitute the acceptor substrate-binding site of the sulphotransferase domain (hNST1) [Kakuta, Sueyoshi, Negishi and Pedersen (1999) J. Biol. Chem. 274, 10673–10676]. When a hexasaccharide model chain is docked into the active site, only a trisaccharide (-IdoA-GlcN-IdoA-) portion interacts directly with the cleft residues: Trp-713, His-716 and His-720 from α helix 6, and Phe-640, Glu-641, Glu-642, Gln-644 and Asn-647 from random coil (residues 640–647). Mutation of these residues either abolishes or greatly reduces hNST1 activity. Glu-642 may play the critical role of catalytic base in the sulphuryl group transfer reaction, as indicated by its hydrogen-bonding distance to the NH3+ group of the glucosamine moiety in the model and by mutational data.

scholarly journals Structural and Technological Approach to Reveal the Role of the Lipid Phase in the Formation of Soy Emulsion Gels with Chia Oil

Gels ◽  
2021 ◽  
Vol 7 (2) ◽  
pp. 48
Author(s):  
Ana M. Herrero ◽  
Claudia Ruiz-Capillas
Keyword(s):  
Raman Spectroscopy ◽  
Structural Properties ◽  
Structural Changes ◽  
Protein Secondary Structure ◽  
Lipid Phase ◽  
Α Helix ◽  
Β Sheet ◽  
Shed Light ◽  
Chia Oil

Considerable attention has been paid to emulsion gels (EGs) in recent years due to their interesting applications in food. The aim of this work is to shed light on the role played by chia oil in the technological and structural properties of EGs made from soy protein isolates (SPI) and alginate. Two systems were studied: oil-free SPI gels (SPI/G) and the corresponding SPI EGs (SPI/EG) that contain chia oil. The proximate composition, technological properties (syneresis, pH, color and texture) and structural properties using Raman spectroscopy were determined for SPI/G and SPI/EG. No noticeable (p > 0.05) syneresis was observed in either sample. The pH values were similar (p > 0.05) for SPI/G and SPI/EG, but their texture and color differed significantly depending on the presence of chia oil. SPI/EG featured significantly lower redness and more lightness and yellowness and exhibited greater puncture and gel strengths than SPI/G. Raman spectroscopy revealed significant changes in the protein secondary structure, i.e., higher (p < 0.05) α-helix and lower (p < 0.05) β-sheet, turn and unordered structures, after the incorporation of chia oil to form the corresponding SPI/EG. Apparently, there is a correlation between these structural changes and the textural modifications observed.

Exploring the Role of the Central Carbide of the Nitrogenase Active-Site FeMo-cofactor through Targeted 13C Labeling and ENDOR Spectroscopy

2021 ◽  
Author(s):  
Ana Pérez-González ◽  
Zhi-Yong Yang ◽  
Dmitriy A. Lukoyanov ◽  
Dennis R. Dean ◽  
Lance C. Seefeldt ◽  
...  
Keyword(s):  
Active Site ◽  
13C Labeling ◽  
Endor Spectroscopy ◽  
Femo Cofactor

scholarly journals Role of Branched-Chain Fatty Acids in pH Stress Tolerance in Listeria monocytogenes

2006 ◽  
Vol 73 (3) ◽  
pp. 997-1001 ◽  
Author(s):  
Efstathios S. Giotis ◽  
David A. McDowell ◽  
Ian S. Blair ◽  
Brian J. Wilkinson
Keyword(s):  
Fatty Acids ◽  
Listeria Monocytogenes ◽  
Stress Tolerance ◽  
Ph Sensitive ◽  
Branched Chain Fatty Acids ◽  
Alkaline Conditions ◽  
Branched Chain ◽  
Ph Adaptation ◽  
Chain Fatty Acids

ABSTRACT In alkaline conditions, Listeria monocytogenes cells develop higher proportions of branched-chain fatty acids (FAs), including more anteiso forms. In acid conditions, the opposite occurs. Reduced growth of pH-sensitive mutants at adverse pH (5.0/9.0) was alleviated by the addition of 2-methylbutyrate (an anteiso-FA precursor), suggesting that anteiso-FAs are important in adaptation to adverse pH. The balance between anteiso- and iso-FAs may be more important than changes in the amounts and/or degrees of saturation of FAs in pH adaptation.

scholarly journals The Role of Active Site Arginines of Sorghum NADP-Malate Dehydrogenase in Thioredoxin-dependent Activation and Activity

2000 ◽  
Vol 275 (46) ◽  
pp. 35792-35798 ◽  
Author(s):  
Isabelle Schepens ◽  
Eric Ruelland ◽  
Myroslawa Miginiac-Maslow ◽  
Pierre Le Maréchal ◽  
Paulette Decottignies
Keyword(s):  
Malate Dehydrogenase ◽  
Active Site
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