Improving Both the Thermostability and Catalytic Efficiency of Phospholipase D from Moritella sp. JT01 through Disulfide Bond Engineering Strategy

Li, Lilang; Mao, Xuejing; Deng, Fuli; Wang, Yonghua; Wang, Fanghua

doi:10.3390/ijms231911319

Cited by 4 publications

(1 citation statement)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Most GH7 endoglucanases with a long chain loop B3 have poor thermostability [21][22][23], suggesting the possible role of loop B3 in structural rigidity. Moreover, disul de bonds can improve the protein rigidity and thermostability in a uctuating cellular environment [24][25][26] or contribute to protein activity by stabilizing active protein conformations [27][28][29]. For example, the introduction of disul de bonds into the unstable exible region or the Nterminus enhanced the activity half-life and melting temperature of xylanases [30].…”

Section: Introductionmentioning

confidence: 99%

Modulation of the catalytic activity and thermostability of a highly thermostable GH7 endoglucanase by engineering the key loop B3

Zheng

Jun-zhao

Qing-yang

et al. 2023

Preprint

View full text Add to dashboard Cite

Background The cellulases of glycoside hydrolase family 7 (GH7) are confined into two main types, endoglucanase and cellobiohydrolase, based on their subtle differences in loop structures. In the viewpoint of evolution, the loop regions of GH7 cellulases exhibit a more pronounced effect on enzyme properties. Results A thermophilic endoglucanase of GH7, TtCel7, having a long 18 amino acid loop B3 was identified in Thermothelomyces thermophilus ATCC 42464. It was successfully obtained with heterologous expression and then purified for activity assays. The recombinant TtCel7 was distinguished for the excellent thermostability at 90°C (> 30% residual activity after 1-h incubation). When truncated the loop B3 or mutated C220A to remove the disulfide bond on loop B3, both the TtCel7 variants showed decreased catalytic efficiency, but the ∆B3 showed improved thermostability, retaining higher residual activities (9–44%) at 70–90°C than the wild type. Based on the molecular dynamics (MD) simulation analysis, both the loops B1 and A3 of ∆B3 swing toward the catalytic center, which contributes to the reduced cleft space and more rigid structure; instead, the structural rigidity of C220A was decreased as an α-helix was introduced into the loop B3 due to the deletion of disulfide bond. Conclusions Two structural elements related to catalysis and thermostability of GH7 cellulases were identified in this study through structure-directed enzyme modulation. Of them, the loop B3 of TtCel7 possibly stretches the catalytic pocket, making the catalytic tunnel more open and the protein structure more flexible for efficient catalysis. Additionally, the disulfide bond in loop B3 stabilizes the loop structure and keeps it in a highly active and stable state. This strategy casts an insight into the engineering of GH7 endoglucanases for potential commercialization.

show abstract