Probing Substrate Promiscuity of Amylosucrase from Neisseria polysaccharea

Abstract

The amylosucrase from Neisseria polysaccharea (NpAS) naturally catalyzes the synthesis of a variety of products from sucrose and shows signs of plasticity of its active site. To explore further this promiscuity, the tolerance of amylosucrase towards different donor and acceptor substrates was investigated. The selection of alternate donor substrates was first made on the basis of preliminary molecular modeling studies. From 11 potential donors harboring selective derivatizations that were experimentally evaluated, only p‐nitrophenyl‐α‐D‐glucopyranoside was used by the wild‐type enzyme, and this underlines the high specificity of the −1 subsite of NpAS for glucosyl donor substrates. The acceptor substrate promiscuity was further explored by screening 20 hydroxylated molecules, including D‐ and L‐monosaccharides as well as polyols. With the exception of one compound, all were successfully glucosylated, and this showcases the tremendous plasticity of the +1 subsite of NpAS, which is responsible for acceptor recognition. The products obtained from the transglucosylation reactions of three selected acceptors were characterized, and they revealed original structures and enzyme enantiopreference, which were more particularly analyzed by in silico docking analyses.

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

“…Three arginine residues (Arg 226 , Arg 415 and Arg 446 ), located in positive subsites + 2/ + 3, + 4 and + 1 respectively, are particularly important in the transglucosylation reaction, since these play a crucial role in the docking and positioning of acceptors [114,115]. This revealed that, despite the fact that AS exhibits slow rates, the D-glucosyl is specifically recognized by a complex network of interactions [117]. Although quite impressive increases in transglucosylation were achieved (395-fold increase), which were accompanied by decreased apparent K m values, no evidence of significant structural changes that would alter sucrose binding was detected [116].…”

Biotransformation of phloretin by amylosucrase yields three novel dihydrochalcone glucosides

“…They are the first reported phloretin glycosides that possess an ␣ sugar-aglycone linkage. Acceptors that previously have been shown to be glucosylated by Ams enzymes are sugars (Daudé et al, 2013;Schneider et al, 2010) and sugar alcohols (Daudé et al, 2013), hydroquinone (Seo et al, 2012), catechin (Cho et al, 2011), baicalein (Kim et al, 2014), arbutin (Seo et al, 2009), salicin ) and piceid . Therefore, the results described here establish, for the first time, the acceptance of unglycosylated as well as glycosylated chalcone-type compounds as substrates for Glc transfer by a non-Leloir glycosyltransferase.…”

Characterization and mechanism insight of accelerated catalytic promiscuity of Sulfolobus tokodaii (ST0779) peptidase for aldol addition reaction

“…The hydrolase fold includes lipase, esterase, protease/peptidase, and dehalogenase etc. (López-Iglesias et al 2011;Sharma et al 2011;Wu et al 2006Wu et al , 2010Pazmiño et al 2007;Gotor-Fernández et al 2006;Dean et al 2007;Lou et al 2008;Li et al 2008;Branneby et al 2004;Nardini and Dijkstra 1999;Yuryev et al 2010;Daudé et al 2013;Wang et al 2014;Busto et al 2010;Kapoor and Gupta 2012), which Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6758-z) contains supplementary material, which is available to authorized users. has been widely identified as an important source of the enzymes that have promiscuous activities to catalyze a variety of C-C and C-N formation reactions (Hult and Berglund 2007;López-Iglesias et al 2011;Sharma et al 2011;Wu et al 2006;Pazmiño et al 2007;Gotor-Fernández et al 2006;Dean et al 2007;Lou et al 2008;Li et al 2008;Branneby et al 2004;Nardini and Dijkstra 1999;Yuryev et al 2010;Daudé et al 2013;Wang et al 2014).…”