Biochemical Characterization of Recombinant UDP-Glucose:Sterol 3-O-Glycosyltransferase from Micromonospora rhodorangea ATCC 31603 and Enzymatic Biosynthesis of Sterol-3-O-��-Glucosides

Abstract

A uridine diphosphate-glucose:sterol glycosyltransferase-encoding gene was isolated and cloned from the established fosmid library of Micromonospora rhodorangea ATCC 27932 that usually produces the aminoglycoside antibiotic geneticin. The gene consists of 1,185 base pairs and encodes a 41.4 kDa protein, which was heterologously expressed in Escherichia coli BL21(DE3). In silico analyses of the deduced gene product suggested that it is a member of the family 1 glycosyltransferases. The recombinant protein MrSGT was able to catalyze the transfer of a glucosyl moiety onto the C-3 hydroxy function in sterols (β-sitosterol, campesterol, and cholesterol), resulting in the corresponding steryl glucosides (β-sitosterol-3-O-β-D-glucoside, campesterol-3-O-β-D-glucoside, and cholesterol-3-O-β-D-glucoside). This enzyme prefers phytosterols to cholesterol, and also shows substrate flexibility to some extent, in that it could recognize a number of acceptor substrates.

Kinetic studies on recombinant UDP-glucose: sterol 3-O-β-glycosyltransferase from Micromonospora rhodorangea and its bioconversion potential

“…The purified His-tagged recombinant MrSGT was prepared as previously described in our recent publication (Hoang et al 2016 ). The authentic sterols such as β-sitosterol, campesterol and cholesterol (Fig.…”

“…Qualitative analyses of the sterols and sterol glucosides (including β-sitosterol-3- O -β- d -glucoside, campesterol-3- O -β- d -glucoside and cholesterol-3- O -β- d -glucoside) obtained from the in vitro MrSGT reactions were conducted by HPLC-tandem mass spectrometry (MS/MS; ThermoFinnigan, San Jose, CA, USA) as described before (Hoang et al 2016 ). In brief, isocratic elution (methanol:acetonitrile:water:formic acid = 45:40:14.8:0.2 [v/v/v/v]) was performed on an Acquity CSH C 18 reversed-phase column (Waters, Milford, MA, USA; 2.1 × 50 mm, 1.7 μm) at a flow rate of 120 μL/min.…”

“…Recently, we isolated and sequenced an open reading frame expected to encode SGT, from the fosmid libraries of Micromonospora rhodorangea ATCC 27932 (deposited as GenBank Accession Number KT983252), using specific degenerate PCR primers (Hoang et al 2016 ). The recombinant gene product, MrSGT, expressed in E. coli BL21(DE3) as a His-tagged protein, was shown to be capable of transferring a glucosyl moiety from UDP-Glc to the free C-3 hydroxyl position of phytosterols (including β-sitosterol and campesterol) and cholesterol.…”

Steryl Glycosides in Fungal Pathogenesis: An Understudied Immunomodulatory Adjuvant

“…There have been several biotechnological advancements using the SG metabolism enzymes, but not SGs themselves. These advancements span the food industry, agriculture, medicinal, and crystallization assays [6,72,73,80,[85][86][87]. Three important studies showed the importance and uses of SGs in these fields.…”

“…First, since SGs are made in such small amounts in cells, a means to mass produce will possibly be needed in the future, especially if our hypothesis regarding SGs as an immunoadjuvant holds true. One 2016 study reported the biochemical characterization of a steryl glycosyltransferase enzyme from the bacteria Micromonospora rhodorangea ATCC 31,603 [85]. This was a unique characterization since this SGT enzyme preferred phytosterols (plant sterols including sitosterol, stigmasterol, and campesterol) to cholesterol when making SGs.…”