A GH13 glycoside phosphorylase with unknown substrate specificity from Corallococcus coralloides

Abstract

Glycoside phosphorylases in subfamily GH13_18 of the carbohydrate-active enzyme database CAZy catalyse the reversible phosphorolysis of α-glycosidic bonds. They contribute to a more energy-efficient metabolism in vivo, and can be applied for the synthesis of valuable glucosides, sugars or sugar phosphates in vitro. Continuing our efforts to uncover new phosphorylase specificities, we identified an enzyme from the myxobacterium Corallococcus coralloides DSM 2259 that does not feature the signature sequence patterns of previously characterised phosphorylases. The enzyme was recombinantly expressed and subjected to substrate screening. Although it was confirmed that the Corallococcus phosphorylase does not have the same substrate specificity as other phoshorylases from subfamily GH13_18, its true natural substrate remains a mystery for now. Myxobacteria have been widely investigated as producers of numerous bioactive secondary metabolites for decades, but little research has been conducted on myxobacterial proteins. The present study exemplifies the untapped metabolic activities and functional diversity that these fascinating organisms may have left to show.

Disaccharide phosphorylases: Structure, catalytic mechanisms and directed evolution

“…All of the DSPs except for SPs, which have been found in nature, show strict stereo- and regiospecificities, for phosphorolysis they prefer a single disaccharide in the forward direction, and in reverse reactions, the high specificity of the enzymes is reflected in a preference for both the donor and acceptor, in addition to the regioselectivity of the synthesized glycosidic bond [ 50 , 51 ]. The dual donor and acceptor specificity is given by the structure of the enzyme active site that recognizes the substrates via not only stacking interactions but also through a network of hydrogen bonds and van der Waals interactions [ [52] , [53] , [54] ].…”

Sucrose Phosphorylase and Related Enzymes in Glycoside Hydrolase Family 13: Discovery, Application and Engineering

“…An isolated clade in the phylogenetic tree of subfamily GH13_18 contains a few enzymes from Corallococcus species that do not contain the signature sequence patterns of the characterised phosphorylases, with a GEXRPYE motif in loop A and an AETD motif in the loop that holds the catalytic acid/base residue ( Figure 2, Figure S1) [20]. Despite an extensive screening of possible substrates, their function remains obscure at this time.…”