Substrate-Binding Mode and Intermediate-Product Distribution Coguided Protein Design of Alginate Lyase AlyF for Altered End-Product Distribution

2021 ◽  
Author(s):  
Keke Zhang ◽  
Yan Yang ◽  
Weidong Wang ◽  
Weizhi Liu ◽  
Qianqian Lyu
Keyword(s):  
Protein Design ◽  
Intermediate Product ◽  
Substrate Binding ◽  
Alginate Lyase ◽  
Binding Mode ◽  
Product Distribution

scholarly journals The substrate-binding cap of the UDP-diacylglucosamine pyrophosphatase LpxH is highly flexible, enabling facile substrate binding and product release

2018 ◽  
Vol 293 (21) ◽  
pp. 7969-7981 ◽  
Author(s):  
Thomas E. Bohl ◽  
Pek Ieong ◽  
John K. Lee ◽  
Thomas Lee ◽  
Jayakanth Kankanala ◽  
...  
Keyword(s):  
Rational Design ◽  
Substrate Binding ◽  
Flexible Substrate ◽  
Binding Mode ◽  
Antibiotic Development ◽  
Product Release ◽  
Outer Leaflet ◽  
Hydrogen Deuterium ◽  
Dynamics Simulations ◽  
Secondary Membrane

Gram-negative bacteria are surrounded by a secondary membrane of which the outer leaflet is composed of the glycolipid lipopolysaccharide (LPS), which guards against hydrophobic toxins, including many antibiotics. Therefore, LPS synthesis in bacteria is an attractive target for antibiotic development. LpxH is a pyrophosphatase involved in LPS synthesis, and previous structures revealed that LpxH has a helical cap that binds its lipid substrates. Here, crystallography and hydrogen–deuterium exchange MS provided evidence for a highly flexible substrate-binding cap in LpxH. Furthermore, molecular dynamics simulations disclosed how the helices of the cap may open to allow substrate entry. The predicted opening mechanism was supported by activity assays of LpxH variants. Finally, we confirmed biochemically that LpxH is inhibited by a previously identified antibacterial compound, determined the potency of this inhibitor, and modeled its binding mode in the LpxH active site. In summary, our work provides evidence that the substrate-binding cap of LpxH is highly dynamic, thus allowing for facile substrate binding and product release between the capping helices. Our results also pave the way for the rational design of more potent LpxH inhibitors.

scholarly journals Substitution of one calcium-binding amino acid strengthens substrate binding in a thermophilic alginate lyase

FEBS Letters ◽  
2018 ◽  
Vol 592 (3) ◽  
pp. 369-379 ◽  
Author(s):  
Bing Wang ◽  
Shi-Qi Ji ◽  
Xiao-Qing Ma ◽  
Ming Lu ◽  
Lu-Shan Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Calcium Binding ◽  
Substrate Binding ◽  
Alginate Lyase

scholarly journals Rigid fusions of designed helical repeat binding proteins efficiently protect a binding surface from crystal contacts

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Patrick Ernst ◽  
Annemarie Honegger ◽  
Floor van der Valk ◽  
Christina Ewald ◽  
Peer R. E. Mittl ◽  
...  
Keyword(s):  
Protein Design ◽  
Binding Proteins ◽  
Peptide Binding ◽  
Binding Mode ◽  
Equilibrium Structure ◽  
Repeat Proteins ◽  
Binding Surface ◽  
Crystal Contacts ◽  
Armadillo Repeat ◽  
Ankyrin Repeat Proteins

Abstract Designed armadillo repeat proteins (dArmRPs) bind extended peptides in a modular way. The consensus version recognises alternating arginines and lysines, with one dipeptide per repeat. For generating new binding specificities, the rapid and robust analysis by crystallography is key. Yet, we have previously found that crystal contacts can strongly influence this analysis, by displacing the peptide and potentially distorting the overall geometry of the scaffold. Therefore, we now used protein design to minimise these effects and expand the previously described concept of shared helices to rigidly connect dArmRPs and designed ankyrin repeat proteins (DARPins), which serve as a crystallisation chaperone. To shield the peptide-binding surface from crystal contacts, we rigidly fused two DARPins to the N- and C-terminal repeat of the dArmRP and linked the two DARPins by a disulfide bond. In this ring-like structure, peptide binding, on the inside of the ring, is very regular and undistorted, highlighting the truly modular binding mode. Thus, protein design was utilised to construct a well crystallising scaffold that prevents interference from crystal contacts with peptide binding and maintains the equilibrium structure of the dArmRP. Rigid DARPin-dArmRPs fusions will also be useful when chimeric binding proteins with predefined geometries are required.

Predictable Selectivity in Remote C−H Oxidation of Steroids: Analysis of Substrate Binding Mode

2020 ◽  
Vol 59 (31) ◽  
pp. 12703-12708 ◽  
Author(s):  
Giorgio Olivo ◽  
Giorgio Capocasa ◽  
Barbara Ticconi ◽  
Osvaldo Lanzalunga ◽  
Stefano Di Stefano ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Binding Mode

Fine-tuning of the substrate binding mode to enhance the catalytic efficiency of an ortho-haloacetophenone-specific carbonyl reductase

2020 ◽  
Vol 10 (8) ◽  
pp. 2462-2472
Author(s):  
Aipeng Li ◽  
Xue Li ◽  
Wei Pang ◽  
Qing Tian ◽  
Ting Wang ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Catalytic Efficiency ◽  
Binding Mode ◽  
Carbonyl Reductase ◽  
Fine Tuning

Fine-tuning of the substrate binding mode was successfully applied for enhancing the catalytic efficiency of an ortho-haloacetophenone-specific carbonyl reductase.

Structure of a gut microbial diltiazem-metabolizing enzyme suggests possible substrate binding mode

2020 ◽  
Vol 527 (3) ◽  
pp. 799-804
Author(s):  
Shuyu Zhou ◽  
Tzu-Ping Ko ◽  
Jian-Wen Huang ◽  
Weidong Liu ◽  
Yingying Zheng ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Binding Mode ◽  
Metabolizing Enzyme

A Nitrogenase Cluster Model [Fe8S6O] with an Oxygen Unsymmetrically Bridging Two Proto-Fe4S3 Cubes: Relevancy to the Substrate Binding Mode of the FeMo Cofactor

2012 ◽  
Vol 51 (21) ◽  
pp. 11217-11219 ◽  
Author(s):  
Shun Ohta ◽  
Yasuhiro Ohki ◽  
Takayoshi Hashimoto ◽  
Roger E. Cramer ◽  
Kazuyuki Tatsumi
Keyword(s):  
Cluster Model ◽  
Substrate Binding ◽  
Binding Mode ◽  
Femo Cofactor
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