Thermal Sensitivity of the Enzymatic Activity of β-Glucosidase: Simulations Lend Mechanistic Insights into Experimental Observations

Sahu, Sneha; Ghosh, Sayani; Sinha, Sushant K.; Datta, Supratim; Sengupta, Neelanjana

doi:10.1021/acs.biochem.3c00387

Cited by 1 publication

(2 citation statements)

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“…The enhanced conformational flexibility around the active site, as indicated by its RMSF value, due to the Q214S/A264S/F344I mutations, would assist in reshaping the dynamic conformational landscape away from inactive substates that might be dominant at low temperatures (Nagel et al, 2011). The inactive substates may differ from the active substates in key molecular features, such as hydrogen bonding patterns (Sahu et al, 2023). Hence, high conformational flexibility around the active site may facilitate substate exchanges and thus provide an entropic advantage in active site-substrate interaction (Sahu et al, 2023).…”

Section: Discussionmentioning

confidence: 99%

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Hotspot Wizard‐informed engineering of a hyperthermophilic β‐glucosidase for enhanced enzyme activity at low temperatures

Erkanli,

El‐Halabi,

Kang

et al. 2024

Biotech & Bioengineering

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Hyperthermophilic enzymes serve as an important source of industrial enzymes due to their high thermostability. Unfortunately, most hyperthermophilic enzymes suffer from reduced activity at low temperatures (e.g., ambient temperature), limiting their applicability. In addition, evolving hyperthermophilic enzymes to increase low temperature activity without compromising other desired properties is generally difficult. In the current study, a variant of β‐glucosidase from Pyrococcus furiosus (PfBGL) was engineered to enhance enzyme activity at low temperatures through the construction of a saturation mutagenesis library guided by the HotSpot Wizard analysis, followed by its screening for activity and thermostability. From this library construction and screening, one PfBGL mutant, PfBGL‐A4 containing Q214S/A264S/F344I mutations, showed an over twofold increase in β‐glucosidase activity at 25 and 50°C compared to the wild type, without compromising high‐temperature activity, thermostability and substrate specificity. Our experimental and computational characterizations suggest that the findings with PfBGL‐A4 may be due to the elevation of local conformational flexibility around the active site, while slightly compacting the global protein structure. This study showcases the potential of HotSpot Wizard‐informed engineering of hyperthermophilic enzymes and underscores the interplays among temperature, enzyme activity, and conformational flexibility in these enzymes.

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Section: Discussionmentioning

confidence: 99%

“…The inactive substates may differ from the active substates in key molecular features, such as hydrogen bonding patterns (Sahu et al, 2023). Hence, high conformational flexibility around the active site may facilitate substate exchanges and thus provide an entropic advantage in active site-substrate interaction (Sahu et al, 2023).…”

Section: Discussionmentioning

confidence: 99%