IDENTIFICATION OF A NOVEL CELLULOSE-BINDING DOMAIN WITHIN THE MULTIDOMAIN 120-KDA XYLANASE XYNA OF THE HYPERTHERMOPHILIC BACTERIUM THERMOTOGA-MARITIMA

A segment of Thermotoga maritima strain MSB8 chromosomal DNA was isola ted which encodes an endo-1,4-beta-D-xylanase, and the nucleotide sequ ence of the xylanase gene, designated xynA, was determined, With a hal f-life of about 40 min at 90 degrees C at the optimal pH of 6.2, purif ied recombinant XynA is one of the most thermostable xylanases known. XynA is a 1059-amino-acid (similar to 120kDa) modular enzyme composed of an N-terminal signal peptide and five domains, in the order A1-A2-B -C1-C2. By comparison with other xylanases of family 10 of glycosyl hy drolases, the central similar to 340-amino-acid part (domain B) of Xyn A represents the catalytic domain. The N-terminal similar to 150-amino -acid repeated domains (A1-A2) have no significant similarity to the C -terminal similar to 170-amino-acid repeated domains (C1-C2). Cellulos e binding studies with truncated XynA derivatives and hybrid proteins indicated that the C-terminal repeated domains mediate the binding of XynA to microcrystalline cellulose and that C2 alone can also promote cellulose binding. C1 and C2 did not share amino acid sequence similar ity with any other known cellulose binding domain (CBD) and thus are C BDs of a novel type, Structurally related protein segments which are p robably also Cans were found in other multidomain xylanolytic enzymes. Deletion of the N-terminal repeated domains or of all the non-catalyt ic domains resulted in substantially reduced thermostability while a t runcated xylanase derivative lacking the C-terminal tandem repeat was as thermostable as the full-length enzyme. It is argued that the multi domain organization of some enzymes may be one of the strategies adopt ed by thermophiles to protect their proteins against thermal denaturat ion.