Characterization of the First Bacterial and Thermostable GDP-Mannose 3,5-Epimerase

Abstract

GDP-mannose 3,5-epimerase (GM35E) catalyzes the conversion of GDP-mannose towards GDP-l-galactose and GDP-l-gulose. Although this reaction represents one of the few enzymatic routes towards the production of l-sugars and derivatives, it has not yet been exploited for that purpose. One of the reasons is that so far only GM35Es from plants have been characterized, yielding biocatalysts that are relatively unstable and difficult to express heterologously. Through the mining of sequence databases, we succeeded in identifying a promising bacterial homologue. The gene from the thermophilic organism Methylacidiphilum fumariolicum was codon optimized for expression in Escherichia coli, resulting in the production of 40 mg/L of recombinant protein. The enzyme was found to act as a self-sufficient GM35E, performing three chemical reactions in the same active site. Furthermore, the biocatalyst was highly stable at temperatures up to 55 °C, making it well suited for the synthesis of new carbohydrate products with application in the pharma industry.

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

“…The enzyme is also of interest for the industrial production of such sugars. [232] GME exists as ah omodimer ande ach monomer contains aR ossmann fold domain,w hich binds the NAD + cofactort ightly,a nd as ubstrate-binding domain, which is primarily helical with two short parallel b-sheets ando ne antiparallel b-sheet. The mechanism of the epimerization reactionc atalyzed by GME is quite complex and involves oxidation of the C4 hydroxyl to ak etone by an NAD + cofactor,w hich decreases the pK a of the adjacent CÀHb onds at C3 and C5 to facilitate deprotonation.…”

GDP-altrose as novel product of GDP-mannose 3,5-epimerase: Revisiting its reaction mechanism

“…His 6 -tagged AtGM35E, MfGM35E and variants thereof were recombinantly expressed and purified by Ni-NTA chromatography according to the protocol previously described by Gevaert et al [10].…”

“…Due to the absence of standards for the GDP-products, this study analyzed the hydrolyzed reaction mixture which yields the respective free monosaccharides of which standards are readily available. The epimerization reaction was performed with both AtGM35E and the bacterial GM35E from Methylacidiphilum fumariolicum (MfGM35E) [10]. The chromatographic analysis yielded four peaks, corresponding to mannose, L-galactose, L-gulose and altrose ( Fig.…”

“…Although S115 is also part of the catalytic triad, it was included in our analysis because mutagenesis of the respective residue in Gal4E was found to have no significant effect on the activity [26]. Furthermore, a function has already been assigned to residue R281, namely the stabilization of the thiolate form of the catalytic cysteine [9,10]. The other three residues (M74, T182 and E144) displayed a very high conservation among GM35E sequences (86-100%), indicating that they also play an important role (Fig.…”

“…A remarkable representative of the carbohydrate epimerase family 1 (CEP1) is GDP-mannose 3,5-epimerase (GM35E) [9,10]. This biocatalyst stands out as it is one of the few, and the only one in the CEP1 family, that is able to catalyze epimerization at two stereocenters.…”