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Molecular Docking and Kinetic Studies of the A226N Mutant of Deinococcus geothermalis Amylosucrase with Enhanced Transglucosylation Activity

Journal of Microbiology and Biotechnology 2020년 30권 9호 p.1436 ~ 1442
홍승표, Siziya Inonge Noni, 서명지, 박천석, 서동호,
소속 상세정보
홍승표 ( Hong Seung-Pyo ) - Korea Food Research Institute
 ( Siziya Inonge Noni ) - Jeonbuk National University College of Agriculture and Life Sciences Department of Food Science and Technology
서명지 ( Seo Myung-Ji ) - Incheon National University Division of Bioengineering
박천석 ( Park Cheon-Seok ) - Kyung Hee University Graduate School of Biotechnology
서동호 ( Seo Dong-Ho ) - Jeonbuk National University College of Agriculture and Life Sciences Department of Food Science and Technology

Abstract


Amylosucrase (ASase, E.C. 2.4.1.4) is capable of efficient glucose transfer from sucrose, acting as the sole donor molecule, to various functional acceptor compounds, such as polyphenols and flavonoids. An ASase variant from Deinococcus geothermalis, in which the 226th alanine is replaced with asparagine (DgAS-A226N), shows increased polymerization activity due to changes in the flexibility of the loop near the active site. In this study, we further investigated how the mutation modulates the enzymatic activity of DgAS using molecular dynamics and docking simulations to evaluate interactions between the enzyme and phenolic compounds. The computational analysis revealed that the A226N mutation could induce and stabilize structural changes near the substratebinding site to increase glucose transfer efficiency to phenolic compounds. Kinetic parameters of DgAS-A226N and WT DgAS were determined with sucrose and 4-methylumbelliferone (MU) as donor and acceptor molecules, respectively. The kcat/Km value of DgAS-A226N with MU (6.352 mM-1min-1) was significantly higher than that of DgAS (5.296 mM-1min-1). The enzymatic activity was tested with a small phenolic compound, hydroquinone, and there was a 1.4-fold increase in α-arbutin production. From the results of the study, it was concluded that DgAS-A226N has improved acceptor specificity toward small phenolic compounds by way of stabilizing the active conformation of these compounds.

키워드

Transglucsoylation; amylosucrase; Deinococcus geothermalis; 4-Methylumberlliferone; molecular dynamics; ocking simulation

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