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GDP-mannose 3,5-epimerase (GM35E) catalyzes the double epimerization of GDP-mannose to yield GDP- l -galactose. GDP- l -gulose (C5-epimer) has previously been detected as a byproduct of this reaction, indicating that C3,5-epimerization occurs through an initial epimerization at C5. Given these products, GM35E constitutes a valuable bridge between d - and l -hexoses. In order to fully exploit this potential, the enzyme might be subjected to specificity engineering for which profound mechanistic insights are beneficial. Accordingly, this study further elucidated GM35E's reaction mechanism. For the first time, the production of the C3-epimer GDP-altrose was demonstrated, resulting in an adjustment of the acknowledged reaction mechanism. As GM35E converts GDP-mannose to GDP- l -gulose, GDP-altrose and GDP- l -galactose in a 72:4:4:20 ratio, this indicates that the enzyme does not discriminate between the C3 and C5 position as initial epimerization site. This was also confirmed by a structural investigation. Based on a mutational analysis of the active site, residues S115 and R281 were attributed a stabilizing function, which is believed to support the reactivation process of the catalytic residues. This paper eventually reflected on some engineering strategies that aim to change the enzyme towards a single specificity. [ABSTRACT FROM AUTHOR]

C5-epimerases are promising tools for the production of rare l-hexoses from their more common d-counterparts. On that account, UDP-glucuronate 5-epimerase (UGA5E) attracts attention as this enzyme could prove to be useful for the synthesis of UDP-l-iduronate. Interestingly, l-iduronate is known as a precursor for the production of heparin, an effective anticoagulant. To date, the UGA5E specificity has only been detected in rabbit skin extract, and the respective enzyme has not been characterized in detail or even identified at the molecular level. Accordingly, the current work aimed to shed more light on the properties of UGA5E. Therefore, the pool of putative UGA5Es present in the UniProt database was scrutinized and their sequences were clustered in a phylogenetic tree. However, the examination of two of these enzymes revealed that they actually epimerize UDP-glucuronate at the 4- rather than 5-position. Furthermore, in silico analysis indicated that this should be the case for all sequences that are currently annotated as UGA5E and, hence, that such activity has not yet been discovered in nature. The detected l-iduronate synthesis in rabbit skin extract can probably be assigned to the enzyme chondroitin-glucuronate C5-epimerase, which catalyzes the conversion of d-glucuronate to l-iduronate on a polysaccharide level. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cellobiose 2-epimerase from Rhodothermus marinus (RmCE) reversibly converts a glucose residue to a mannose residue at the reducing end of β-1,4-linked oligosaccharides. In this study, the monosaccharide specificity of RmCE has been mapped and the synthesis of d-talose from d-galactose was discovered, a reaction not yet known to occur in nature. Moreover, the conversion is industrially relevant, as talose and its derivatives have been reported to possess important antimicrobial and anti-inflammatory properties. As the enzyme also catalyzes the keto-aldo isomerization of galactose to tagatose as a minor side reaction, the purity of talose was found to decrease over time. After process optimization, 23 g/L of talose could be obtained with a product purity of 86% and a yield of 8.5% (starting from 4 g (24 mmol) of galactose). However, higher purities and concentrations can be reached by decreasing and increasing the reaction time, respectively. In addition, two engineering attempts have also been performed. First, a mutant library of RmCE was created to try and increase the activity on monosaccharide substrates. Next, two residues from RmCE were introduced in the cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) (S99M/Q371F), increasing the kcat twofold. [ABSTRACT FROM AUTHOR]