EXPRESSION IN ESCHERICHIA-COLI AND STRUCTURE OF THE GENE ENCODING 4-ALPHA-GLUCANOTRANSFERASE FROM THERMOTOGA-MARITIMA - CLASSIFICATION OF MALTODEXTRIN GLYCOSYLTRANSFERASES INTO 2 DISTANTLY RELATED ENZYME SUBFAMILIES

4-alpha-Glucanotransferase (GTase) has been partially purified and sep arated from other amylolytic activities present in the crude extract o f the hyperthermophilic bacterium Thermotoga maritima strain MSB8. Und er the growth conditions employed, the enzyme is produced in only very low amounts. Expression of the cloned GTase gene in Escherichia coli and Corynebacterium glutamicum yielded an active, cytoplasmically loca lized enzyme in both mesophilic host organisms. The GTase-encoding seg ment of the T. maritima chromosome was structurally characterized. the mgtA gene has the potential to code for a polypeptide of 441 amino ac ids with a calculated molecular mass of 51,854 Da. Comparative amino a cid sequence analysis carried out with the GTase primary structure ded uced from the nucleotide sequence of the gene revealed that apart from a few short sequence segments, the T. maritima enzyme was non-related to the amylomaltases of E. coli and Streptococcus pneumoniae which wa s surprising since these enzymes catalyse similar reactions. The seque nce alignment results suggest that maltodextrin glycosyltransferases ( MGTases, 4-alpha-glucanotransferases; EC 2.4.1.25) can be classified i nto two groups which share no significant overall sequence similarity. While the amylomaltases of S. pneumoniae and E. coli and potato D-enz yme comprise one group, GTase is the first member of a new MGTase grou p which is more closely related to alpha-amylases, alpha-glucosidases and cyclodextrin glycosyltransferases than to other MGTases. Finally, GTase as well as the other group of MGTases share limited amino acid s equence similarity with short sequence segments conserved in alpha-amy lases and other structurally related enzymes. Thus, the MGTase groups can be regarded as two subfamilies of the large so-called ''alpha-amyl ase enzyme family'' whose members probably all contain a (beta/alpha)( 8) fold as the core super-secondary structural motif.