Characterisation of a novel amylosucrase from Deinococcus radiodurans

The BLAST search for amylosucrases has yielded several gene sequences of putative amylosucrases, however, with various questionable annotations. The putative encoded proteins share 32–48% identity with Neisseria polysaccharea amylosucrase (AS) and contain several amino acid residues proposed to be involved in AS specificity. First, the B‐domains of the putative proteins and AS are highly similar. In addition, they also reveal additional residues between putative β‐strand 7 and α‐helix 7 which could correspond to the AS B′‐domain, which turns the active site into a deep pocket. Finally, conserved Asp and Arg residues could form a salt bridge similar to that found in AS, which is responsible for the glucosyl unit transfer specificity. Among these found genes, locus NP_294657.1 ( dras ) identified in the Deinococcus radiodurans genome was initially annotated as an α‐amylase encoding gene. The putative encoded protein (DRAS) shares 42% identity with N. polysaccharea AS. To investigate the activity of this protein, gene NP_294657.1 was cloned and expressed in Escherichia coli . When acting on sucrose, the pure recombinant enzyme was shown to catalyse insoluble amylose polymer synthesis accompanied by side‐reactions (sucrose hydrolysis, sucrose isomer and soluble maltooligosaccharide formation). Kinetic analyses further showed that DRAS follows a non‐Michaelian behaviour toward sucrose substrate and is activated by glycogen, as is AS. This demonstrates that gene NP_294657.1 encodes an amylosucrase.