Dual Substrate Specificity of the Rutinosidase from Aspergillus niger and the Role of Its Substrate Tunnel

Rutinosidases (α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranosidases, EC 3.2.1.168, CAZy GH5) are diglycosidases that cleave the glycosidic bond between the disaccharide rutinose and the respective aglycone. Similar to many retaining glycosidases, rutinosidases can also transfer the rutinosyl moiety onto acceptors with a free –OH group (so-called transglycosylation). The recombinant rutinosidase from Aspergillus niger (AnRut) is selectively produced in Pichia pastoris. It can catalyze transglycosylation reactions as an unpurified preparation directly from cultivation. This enzyme exhibits catalytic activity towards two substrates; in addition to rutinosidase activity, it also exhibits β-d-glucopyranosidase activity. As a result, new compounds are formed by β-glucosylation or rutinosylation of acceptors such as alcohols or strong inorganic nucleophiles (NaN3). Transglycosylation products with aliphatic aglycones are resistant towards cleavage by rutinosidase, therefore, their side hydrolysis does not occur, allowing higher transglycosylation yields. Fourteen compounds were synthesized by glucosylation or rutinosylation of selected acceptors. The products were isolated and structurally characterized. Interactions between the transglycosylation products and the recombinant AnRut were analyzed by molecular modeling. We revealed the role of a substrate tunnel in the structure of AnRut, which explained the unusual catalytic properties of this glycosidase and its specific transglycosylation potential. AnRut is attractive for biosynthetic applications, especially for the use of inexpensive substrates (rutin and isoquercitrin).

Dual Substrate Specificity of anN-Acetylglucosamine Phosphotransferase System in Clostridium beijerinckii

ABSTRACTThe solventogenic clostridia have a considerable capacity to ferment carbohydrate substrates with the production of acetone and butanol, making them attractive organisms for the conversion of waste materials to valuable products. In common with other anaerobes, the clostridia show a marked dependence on the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) to accumulate sugars and sugar derivatives. In this study, we demonstrate that extracts ofClostridium beijerinckiigrown onN-acetylglucosamine (GlcNAc) exhibit PTS activity for the amino sugar. The PTS encoded by the divergent genescbe4532(encoding the IIC and IIB domains) andcbe4533(encoding a IIA domain) was shown to transport and phosphorylate GlcNAc and also glucose. When the genes were recombined in series under the control of thelacpromoter in pUC18 and transformed into a phosphotransferase mutant (nagE) ofEscherichia colilacking GlcNAc PTS activity, the ability to take up and ferment GlcNAc was restored, and extracts of the transformant showed PEP-dependent phosphorylation of GlcNAc. The gene products also complemented anE. colimutant lacking glucose PTS activity but were unable to complement the same strain for PTS-dependent mannose utilization. Both GlcNAc and glucose induced the expression ofcbe4532andcbe4533inC. beijerinckii, and consistent with this observation, extracts of cells grown on glucose exhibited PTS activity for GlcNAc, and glucose did not strongly repress utilization of GlcNAc by growing cells. On the basis of the phylogeny and function of the encoded PTS, we propose that the genescbe4532andcbe4533should be designatednagEandnagF, respectively.