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

Aipeng Li; Xue Li; Wei Pang; Qing Tian; Ting Wang; Lianbing Zhang

doi:10.1039/c9cy02335f

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

Catalysis Science & Technology ◽

10.1039/c9cy02335f ◽

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.

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Faculty Opinions recommendation of Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1024182.290792 ◽

2005 ◽

Author(s):

Arieh Warshel

Keyword(s):

Active Site ◽

Substrate Binding ◽

Computational Prediction ◽

Binding Mode

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Fine-tuning dirhodium compounds with bridging ligands: Synthesis, structure, catalytic efficiency

Journal of Organometallic Chemistry ◽

10.1016/j.jorganchem.2021.122078 ◽

2021 ◽

Vol 954-955 ◽

pp. 122078

Author(s):

Yangbo Ning ◽

Jiantao Tan ◽

Zhifan Wang ◽

Yuanhua Wang

Keyword(s):

Catalytic Efficiency ◽

Fine Tuning ◽

Bridging Ligands

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Improving the Substrate Affinity and Catalytic Efficiency of β-Glucosidase Bgl3A from Talaromyces leycettanus JCM12802 by Rational Design

Biomolecules ◽

10.3390/biom11121882 ◽

2021 ◽

Vol 11 (12) ◽

pp. 1882

Author(s):

Wei Xia ◽

Yingguo Bai ◽

Pengjun Shi

Keyword(s):

Hydrogen Bonding ◽

Rational Design ◽

Substrate Binding ◽

Enzymatic Saccharification ◽

Catalytic Efficiency ◽

Binding Pocket ◽

Glycoside Hydrolases ◽

Cellulosic Biomass ◽

Substrate Affinity ◽

Hydrogen Bonding Networks

Improving the substrate affinity and catalytic efficiency of β-glucosidase is necessary for better performance in the enzymatic saccharification of cellulosic biomass because of its ability to prevent cellobiose inhibition on cellulases. Bgl3A from Talaromyces leycettanus JCM12802, identified in our previous work, was considered a suitable candidate enzyme for efficient cellulose saccharification with higher catalytic efficiency on the natural substrate cellobiose compared with other β-glucosidase but showed insufficient substrate affinity. In this work, hydrophobic stacking interaction and hydrogen-bonding networks in the active center of Bgl3A were analyzed and rationally designed to strengthen substrate binding. Three vital residues, Met36, Phe66, and Glu168, which were supposed to influence substrate binding by stabilizing adjacent binding site, were chosen for mutagenesis. The results indicated that strengthening the hydrophobic interaction between stacking aromatic residue and the substrate, and stabilizing the hydrogen-bonding networks in the binding pocket could contribute to the stabilized substrate combination. Four dominant mutants, M36E, M36N, F66Y, and E168Q with significantly lower Km values and 1.4–2.3-fold catalytic efficiencies, were obtained. These findings may provide a valuable reference for the design of other β-glucosidases and even glycoside hydrolases.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.002503 ◽

2018 ◽

Vol 293 (21) ◽

pp. 7969-7981 ◽

Cited By ~ 7

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.

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