KnowVolution Campaign of an Aryl Sulfotransferase Increases Activity toward Cellobiose

0497526 - MBÚ 2019 RIV DE eng J - Článek v odborném periodiku
Islam, S. - Laaf, D. - Infanzon, B. - Pelantová, Helena - Davari, M.D. - Jacob, F. - Křen, Vladimír - Elling, L. - Schwaneberg, U.
KnowVolution Campaign of an Aryl Sulfotransferase Increases Activity toward Cellobiose.
Chemistry - A European Journal. Roč. 24, č. 64 (2018), s. 17117-17124. ISSN 0947-6539. E-ISSN 1521-3765
Grant CEP: GA ČR(CZ) GA18-01163S
Institucionální podpora: RVO:61388971
Klíčová slova: aryl sulfotransferase * biocatalyst * directed evolution * protein engineering
Obor OECD: Biochemistry and molecular biology
Impakt faktor: 5.160, rok: 2018

Sulfated polysaccharides such as cellulose can mimic the functionalities of pathophysiologically important glycosaminoglycans. Enzymatic sulfation offers a green chemistry route to selective (mono)sulfation of oligosaccharides (e.g., cellobiose as a building block of cellulose) in aqueous solution, at ambient temperature, and high chemoselectivity. Here, we report the first KnowVolution campaign for the aryl sulfotransferase B (ASTB) from Desulfitobacterium hafniense to advance ASTB toward a synthetically attractive biocatalyst. The generated final recombination variant (ASTB-M5) carries two amino acid substitutions (Leu446Pro and Val579Lys) leading to an up to 7.6-fold increase in specific activity (6.15 U mg(-1)) that was obtained with one round of KnowVolution. Mass spectrometry analysis confirmed a monosulfated product of cellobiose and structure elucidation by NMR confirmed the sulfation at the positions C-3 or C-4 of GlcNAc-linker-tBoc as opposed to the preferred C-6 by chemical means. Computational analysis suggested an important role of Leu446Pro in substrate-binding and recognized Val579Lys as a distal substitution.
Trvalý link: http://hdl.handle.net/11104/0290096