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 separated from other amylolytic activities present in the crude extract of the hyperthermophilic bacterium Thermotoga maritima strain MSB8. Under 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 localized enzyme in both mesophilic host organisms. The GTase-encoding segment of the T. maritima chromosome was structurally characterized. the mgtA gene has the potential to code for a polypeptide of 441 amino acids with a calculated molecular mass of 51,854 Da. Comparative amino acid sequence analysis carried out with the GTase primary structure deduced 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 was surprising since these enzymes catalyse similar reactions. The sequence alignment results suggest that maltodextrin glycosyltransferases (MGTases, 4-alpha-glucanotransferases; EC 2.4.1.25) can be classified into two groups which share no significant overall sequence similarity. While the amylomaltases of S. pneumoniae and E. coli and potato D-enzyme comprise one group, GTase is the first member of a new MGTase group 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 sequence similarity with short sequence segments conserved in alpha-amylases and other structurally related enzymes. Thus, the MGTase groups can be regarded as two subfamilies of the large so-called ''alpha-amylase enzyme family'' whose members probably all contain a (beta/alpha)(8) fold as the core super-secondary structural motif.