Pcal_0976, a pullulanase homologue from Pyrobaculum calidifontis, displays a glycoside hydrolase activity but no pullulanase activity.

In this study, we have cloned and characterized a novel protein, Pcal_0976, annotated as pullulanase in the genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis. Motif search showed two glucodextran_C like domains and a domain of unknown function (DUF4134) in Pcal_0976. Multiple alignment with close homologues demonstrated six stretches of conserved regions. When the gene encoding Pcal_0976 was expressed in Escherichia coli, the recombinant protein was produced in insoluble and inactive form, which was solubilized using guanidine hydrochloride and refolded in an active form in the presence of arginine. Refolded Pcal_0976 displayed hydrolysis of glycogen, dextran, dextrin and starch. No hydrolytic activity was detected against pullulan. These results indicate that Pcal_0976 may not be a pullulanase but a novel glycoside hydrolase. Further studies are needed to establish the role of Pcal_0976 in carbohydrate metabolism in this archaeon. [ABSTRACT FROM AUTHOR]