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 beta-strand 7 and alpha-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 alpha-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.
Pizzut-Serin
Sandra
S
Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, UMR INRA 792, INSA, 135 avenue de Rangueil, 31077 Toulouse Cedex 4, France.
Potocki-Véronèse
Gabrielle
G
van der Veen
Bart A
BA
Albenne
Cécile
C
Monsan
Pierre
P
Remaud-Simeon
Magali
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D034301:Deinococcus / Q000201:enzymology* / Q000235:genetics; D005632:Fructose / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006003:Glycogen / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment
D005632:Fructose; D006003:Glycogen; D005964:Glucosyltransferases; C019700:amylosucrase