Selection and mutational analyses of the substrate interacting residues of a chitinase from Enterobacter cloacae subsp. cloacae (EcChi2) to improve transglycosylation.

Autor: Mallakuntla MK; Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad 50046, Telangana, India., Vaikuntapu PR; ICAR- Directorate of Groundnut Research, Ivnagar Road, Junagadh, Gujarath 362001, India., Bhuvanachandra B; Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad 50046, Telangana, India., Podile AR; Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad 50046, Telangana, India. Electronic address: arpsl@uohyd.ernet.in.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2020 Dec 15; Vol. 165 (Pt B), pp. 2432-2441. Date of Electronic Publication: 2020 Oct 20.
DOI: 10.1016/j.ijbiomac.2020.10.125
Abstrakt: Transglycosylation (TG) by Enterobacter cloacae subsp. cloacae chitinase 2 (EcChi2) has been deciphered by site-directed mutagenesis. EcChi2 originally displayed feeble TG with chitin oligomer with a degree of polymerization (DP4), for a short duration. Based on the 3D modelling and molecular docking analyses, we altered the substrate interactions at the substrate-binding cleft, catalytic center, and catalytic groove of EcChi2 by mutational approach to improve TG. The mutation of W166A and T277A increased TG by EcChi2 and also affected its catalytic efficiency on the polymeric substrates. Whereas, R171A had a drastically decreased hydrolytic activity but, retained TG activity. In the increased hydrolytic activity of the T277A, altered interactions with the substrates played an indirect role in the catalysis. Mutation of the central Asp, in the conserved DxDxE motif, to Ala (D314A) and Asn (D314N) conversion yielded DP5-DP8 TG products. The quantifiable TG products (DP5 and DP6) increased to 8% (D314A) and 7% (D314N), resulting in a hyper-transglycosylating mutant. Mutation of W276A and W398A resulted in the loss of TG activity, indicating that the aromatic residues (W276 and W398) at +1 and +2 subsites are essential for the TG activity of EcChi2.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2020. Published by Elsevier B.V.)
Databáze: MEDLINE
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