Zn2+ stapling of N and C-terminal maintains stability and substrate affinity in GH26 endo-mannanase

Enzyme and Microbial Technology
Gaurav Singh KairaMukesh Kapoor

Abstract

Metal binding sites are present in one-third of proteins and are crucial for biological functions and structural maintenance. GH26 endo-mannanase (ManB-1601) from Bacillus sp. harbors a Zn2+ binding site which connects N (H1, H23) and C (E336)-terminal residues. Present study reveals how native circularization of ManB-1601 through Zn2+ coordination regulates the structure-function. We generated individual Zn2+ coordinating mutants and characterized them using biochemical and biophysical approaches. Contribution of individual Zn2+ coordination towards maintaining ManB-1601 stability and rigidity was in the following order H23>H1 > E336. Elimination of E336 and H23-Zn2+ coordination affected substrate hydrolysis to a greater degree than H1-Zn2+ coordination. Metal quantification of mutant proteins indicated that H23A did not contain Zn2+. Molecular dynamic simulation studies revealed disruption of H23-Zn2+ coordination leads to increased flexibility of N and C-terminal, active site loops and consequent drifting of substrate away from the active site region. Finally, mechanistic understanding on the functioning of Zn2+ site in ManB-1601 is developed wherein 1) H23 by anchoring Zn2+ ion majorly regulates the structure-function prop...Continue Reading

Citations

Oct 10, 2020·Critical Reviews in Biotechnology·Gaurav Singh Kaira, Mukesh Kapoor

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