scholarly journals Transport Efficiency of AtGTR1 Dependents on the Hydrophobicity of Transported Glucosinolates

2021 ◽  
Author(s):  
Yi-Chia Chung ◽  
Hao-Yu Cheng ◽  
Wei-Tung Wang ◽  
Yen-Jui Chang ◽  
Shih-Ming Lin
Keyword(s):  
Regulatory Mechanism ◽  
Binding Pocket ◽  
Substrate Affinity ◽  
Transport Function ◽  
Substrate Binding Pocket ◽  
Transport Efficiency ◽  
Substrate Preferences ◽  
Uptake Assay ◽  
Indole 3 Acetic Acid

Abstract Glucosinolates (GLSs) are a group of secondary metabolites that are involved in the defense of herbivores. In Arabidopsis thaliana, Glucosinolate Transporter 1 (AtGTR1) transports GLSs with high affinity via a proton gradient-driven process. In addition to transporting GLSs, AtGTR1 also transports phytohormones, jasmonic acidisoleucine (JA-Ile), and gibberellin (GA). However, little is known about the mechanisms underlying the broad substrate specificity of AtGTR1. Here, we characterized the substrate preference of AtGTR1 by using a yeast uptake assay, and the results revealed that GLS transport rates are negatively correlated with the hydrophobicity of substrates. Interestingly, the AtGTR1 showed a higher substrate affinity for GLSs with higher hydrophobicity, suggesting a hydrophobic substrate binding pocket. In addition, a competition assay revealed that the presence of JA, salicylic acid (SA), and indole-3-acetic acid (IAA) inhibits the transport of GLSs in yeast, suggesting a potential regulatory mechanism of AtGTR1. To further characterize the functional properties of AtGTR1, mutagenesis experiments confirmed that the conserved EXXEK motif and Arg166 are essential for the GLS transport function. In addition, the purified AtGTR1 adopts a homodimeric conformation, which is possibly regulated by phosphorylation on Thr105. The phosphomimetic mutation, T105D, reduced its protein expression and completely abrogated its GLS transport function, indicating the essential role of phosphorylation on AtGTR1. In summary, this study investigated various factors associated with the GLS transport and increased our knowledge on the substrate preferences of AtGTR1. These findings contribute to understanding how the distribution of defense GLSs is regulated in plants and could be used to improve crop quality in agriculture.

scholarly journals Reprogramming protein kinase substrate specificity through synthetic mutations

2016 ◽  
Author(s):  
Joshua M. Lubner ◽  
George M. Church ◽  
Michael F. Chou ◽  
Daniel Schwartz
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Structure Function ◽  
Binding Pocket ◽  
Missense Mutations ◽  
New Paradigm ◽  
Substrate Binding Pocket ◽  
Kinase Substrate ◽  
Substrate Preferences ◽  
Kinase Specificity

Protein kinase specificity is largely imparted through substrate binding pocket motifs. Missense mutations in these regions are frequently associated with human disease, and in some cases can alter substrate specificity. However, current efforts at decoding the influence of mutations on substrate specificity have been focused on disease-associated mutations. Here, we adapted the Proteomic Peptide Library (ProPeL) approach for determining kinase specificity to the task of exploring structure-function relationships in kinase specificity by interrogating the effects of synthetic mutation. We established a specificity model for the wild-type DYRK1A kinase with unprecedented resolution. Using existing crystallographic and sequence homology data, we rationally designed mutations that precisely reprogrammed the DYRK1A kinase at the P+1 position to mimic the substrate preferences of a related kinase, CK II. This study illustrates a new synthetic biological approach to reprogram kinase specificity by design, and a powerful new paradigm to investigate structure-function relationships underpinning kinase substrate specificity.

scholarly journals Structural insights into the role of the N-terminus in the activation and function of extracellular serine protease from Staphylococcus epidermidis

2020 ◽  
Vol 76 (1) ◽  
pp. 28-40
Author(s):  
Kartik Manne ◽  
Sthanam V. L. Narayana
Keyword(s):  
Serine Protease ◽  
Staphylococcus Epidermidis ◽  
Binding Pocket ◽  
Glutamyl Endopeptidase ◽  
Structural Components ◽  
Substrate Binding Pocket ◽  
N Terminus ◽  
Peptide Segments ◽  
Extracellular Serine Protease

Extracellular serine protease (Esp) from Staphylococcus epidermidis is a glutamyl endopeptidase that inhibits the growth and formation of S. aureus biofilms. Previously, crystal structures of the matured and active Esp have been determined. Interestingly, many of the staphylococcal glutamyl endopeptidase zymogens, including V8 from Staphylococcus aureus and Esp from S. epidermidis, contain unusually long pro-peptide segments; however, their function is not known. With the aim of elucidating the function of these pro-peptide segments, crystal structures of the Esp zymogen (Pro-Esp) and its variants were determined. It was observed that the N-terminus of the Pro-Esp crystal structure is flexible and is not associated with the main body of the enzyme, unlike in the known active Esp structure. In addition, the loops that border the putative substrate-binding pocket of Pro-Esp are flexible and disordered; the structural components that are responsible for enzyme specificity and efficiency in serine proteases are disordered in Pro-Esp. However, the N-terminal locked Pro-Esp variants exhibit a rigid substrate-binding pocket similar to the active Esp structure and regain activity. These structural studies highlight the role of the N-terminus in stabilizing the structural components responsible for the activity and specificity of staphylococcal glutamyl endopeptidases.

Sequence Analysis and Functional Interpretation of α-Amylase Receptor from Anopheles albimanus Using a Molecular Model

2013 ◽  
Vol 798-799 ◽  
pp. 1095-1098
Author(s):  
Shi Ping Shan ◽  
Dong Xia Du ◽  
De Yuan Zhang ◽  
Zhao Hui Guo
Keyword(s):  
Brush Border Membrane ◽  
Molecular Model ◽  
Membrane Vesicle ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Substrate Binding Pocket ◽  
Functional Interpretation ◽  
Three Dimensional Structure

Activated toxins interact with α-amylase receptor on the brush border membrane vesicle (BBMV) of the midgut epithelium, which activates intracellular oncotic pathways and leads to cell death. In order to decipher the mechanism of process how toxins interact with their receptors, it is essential to investigate their three-dimensional structure. The three-dimensional structure of α-amylase was constructed by homology modeling, based on crystal structure ofBacillus cereusoligo-1,6-glucosidase and the model was further evaluated using PROSA energy and ERRAT. The substrate binding pocket responsible for the interactions with toxins was predicted and analyzed, and the important role of binding of toxin to binding pocket on α-amylase was discussed in the aspect of Cry4Ba and Cry11Aa toxicity.

scholarly journals Probing the Role of Sigma π Interaction and Energetics in the Catalytic Efficiency of Endo-1,4-β-Xylanase

2012 ◽  
Vol 78 (24) ◽  
pp. 8817-8821 ◽  
Author(s):  
Raushan Kumar Singh ◽  
Manish Kumar Tiwari ◽  
In-Won Kim ◽  
Zhilei Chen ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acid ◽  
Turnover Rate ◽  
Catalytic Efficiency ◽  
Binding Pocket ◽  
Wild Type ◽  
High Turnover ◽  
Substrate Binding Pocket ◽  
Content Type ◽  
Important Residue

ABSTRACTChaetomium globosumendo-1,4-β-xylanase (XylCg) is distinguished from other xylanases by its high turnover rate (1,860 s−1), the highest ever reported for fungal xylanases. One conserved amino acid, W48, in the substrate binding pocket of wild-type XylCg was identified as an important residue affecting XylCg's catalytic efficiency.

scholarly journals Protonation of Glutamate 208 Induces the Release of Agmatine in an Outward-facing Conformation of an Arginine/Agmatine Antiporter

2011 ◽  
Vol 286 (22) ◽  
pp. 19693-19701 ◽  
Author(s):  
Elia Zomot ◽  
Ivet Bahar
Keyword(s):  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Time Resolved ◽  
Extracellular Region ◽  
Acidic Conditions ◽  
Using Data ◽  
Dynamics Simulations ◽  
The Impact ◽  
First Time

Virulent enteric pathogens have developed several systems that maintain intracellular pH to survive extreme acidic conditions. One such mechanism is the exchange of arginine (Arg+) from the extracellular region with its intracellular decarboxylated form, agmatine (Agm2+). The net result of this process is the export of a virtual proton from the cytoplasm per antiport cycle. Crystal structures of the arginine/agmatine antiporter from Escherichia coli, AdiC, have been recently resolved in both the apo and Arg+-bound outward-facing conformations, which permit us to assess for the first time the time-resolved mechanisms of interactions that enable the specific antiporter functionality of AdiC. Using data from ∼1 μs of molecular dynamics simulations, we show that the protonation of Glu-208 selectively causes the dissociation and release of Agm2+, but not Arg+, to the cell exterior. The impact of Glu-208 protonation is transmitted to the substrate binding pocket via the reorientation of Ile-205 carbonyl group at the irregular portion of transmembrane (TM) helix 6. This effect, which takes place only in the subunits where Agm2+ is released, invites attention to the functional role of the unwound portion of TM helices (TM6 Trp-202–Glu-208 in AdiC) in facilitating substrate translocation, reminiscent of the behavior observed in structurally similar Na+-coupled transporters.

Understanding substrate binding and the role of gatekeeping residues in PigC access tunnels

2021 ◽  
Author(s):  
Stefanie Brands ◽  
Jarno G. Sikkens ◽  
Mehdi D. Davari ◽  
Hannah U. C. Brass ◽  
Andreas S. Klein ◽  
...  
Keyword(s):  
Rational Design ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Short Chain ◽  
Substrate Binding Pocket

Prodigiosin ligase PigC has been engineered by semi-rational design to accept short chain-pyrroles, providing molecular understanding of access tunnels and the substrate-binding pocket.

Structure and function of Plasmodium falciparum malate dehydrogenase: Role of critical amino acids in co-substrate binding pocket

Biochimie ◽  
2009 ◽  
Vol 91 (11-12) ◽  
pp. 1509-1517 ◽  
Author(s):  
Anupam Pradhan ◽  
Abhai K. Tripathi ◽  
Prashant V. Desai ◽  
Prasenjit K. Mukherjee ◽  
Mitchell A. Avery ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasmodium Falciparum ◽  
Malate Dehydrogenase ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Structure And Function ◽  
Substrate Binding Pocket ◽  
And Function

Significant improvement to the catalytic properties of aspartate aminotransferase: Role of hydrophobic and charged residues in the substrate binding pocket

Biochemistry ◽  
1994 ◽  
Vol 33 (1) ◽  
pp. 90-97 ◽  
Author(s):  
Eleonore Koehler ◽  
Mark Seville ◽  
Joachim Jaeger ◽  
Ian Fotheringham ◽  
Michael Hunter ◽  
...  
Keyword(s):  
Aspartate Aminotransferase ◽  
Catalytic Properties ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Charged Residues

scholarly journals Role of a Conserved Membrane Glycine Residue in a Dicarboxylate Transporter from Sinorhizobium meliloti

2006 ◽  
Vol 189 (5) ◽  
pp. 2160-2163 ◽  
Author(s):  
Maria A. Trainer ◽  
Svetlana N. Yurgel ◽  
Michael L. Kahn
Keyword(s):  
Amino Acid ◽  
Sinorhizobium Meliloti ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Significant Activity ◽  
Nitrogen Fixing ◽  
Substrate Binding Pocket ◽  
Glycine Residue

ABSTRACT Nitrogen-fixing rhizobial bacteroids import dicarboxylates by using the DctA transporter. G114 of DctA is highly conserved. A G114D mutant is inactive, but DctA with a small amino acid (G114A) or a helix disrupter (G114P) retains significant activity. G114 probably interacts with other membrane helices in stabilizing a substrate-binding pocket.

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