Biochimica et Biophysica Acta 2011-12-01

Insight into the binding of the wild type and mutated alginate lyase (AlyVI) with its substrate: a computational and experimental study.

Adel Hamza, Yu Lan Piao, Mi-Sun Kim, Cheol Hee Choi, Chang-Guo Zhan, Hoon Cho, Adel Hamza, Yu Lan Piao, Mi-Sun Kim, Cheol Hee Choi, Chang-Guo Zhan, Hoon Cho

Index: Biochim. Biophys. Acta 1814(12) , 1739-47, (2011)

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Abstract

The homology model of the wild type alginate lyase (AlyVI) marine bacterium Vibrio sp. protein, was built using the crystal structure of the Family 7 alginate lyase from Sphingomonas sp. A1. To rationalize the observed structure-affinity relationships of aliginate lyase alyVI with its (GGG) substrate, molecular docking, MD imulations and binding free energy calculations followed by site-directed mutagenesis and alyVI activity assays were carried out. Per-residue decomposition of the (GGG) binding energy revealed that the most important contributions were from polar and charged residues, such as Asn138, Arg143, Asn217, and Lys308, while van der Waals interactions were responsible for binding with the catalytic His200 and Tyr312 residues. The mutants H200A, K308A, Y312A, Y312F, and W165A were found to be inactive or almost inactive. However, the catalytic efficiency (k(cat)/K(m)) of the double mutant L224V/D226G increased by two-fold compared to the wild type enzyme. This first structural model with its substrate binding mode and the agreement with experimental results provide a suitable base for the future rational design of new mutated alyVI structures with improved catalytic activity.Copyright © 2011 Elsevier B.V. All rights reserved.