Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto-oligosaccharides

A novel enzyme acting on starch and malto-oligosaccharides was identified a nd characterised. The non-hydrolytic enzyme, designated maltosyltransferase (MTase), of the hyperthermophilic bacterium Thermotoga maritima MSB8 dispr oportionates malto-oligosaccharides via glycosyl transfer reactions. The en zyme has a unique transfer specificity strictly confined to the transfer of maltosyl units. Incubation of MTase with starch or its constituents, i.e. amylose and amylopectin, led to the formation of a set of multiples of malt ose (i.e. maltose, maltotetraose, maltohexaose etc.). Malto-oligosaccharide s with a degree of polymerization (DP) X were disporportionated to products with a DP of X +/- 2n (with X greater than or equal to 3 and n = 0, 1,2,.. .). Maximum activity in a 10-min assay was recorded at pH 6.5 and 85-90 deg rees C. The enzyme displayed extraordinary resistance to thermal inactivati on. For example, at 90, 85, and 70 degrees C (pH 6.5, 0.34 mg ml(-1) protei n), MTase half-lives of about 2.5 h, 17 h, and 21 days, respectively, were recorded. The gene for MTase, designated mmtA, was isolated from a gene library of T. maritima strain MSB8. Analysis of the MTase primary structure as deduced f rom the nucleotide sequence of mmtA revealed that the enzyme is not closely related to known protein sequences. However, low-level local similarity be tween MTase and the alpha-amylase enzyme family (glycosyl hydrolase family 13) was detected, including conserved acidic residues essential for catalys is. Therefore, MTase should be assigned to this Family. Based on detailed s equence analyses and comparison with amylolytic enzymes of known crystal st ructure we propose that MTase contains a (beta/alpha)(8)-fold as the core s upersecondary structure which is typical for the alpha-amylase family. On t he other hand, MTase is unique in that it lacks several residues highly con served throughout this family. Also, MTase possesses an extraordinarily lar ge domain B (a domain typical for the alpha-amylase family, inserted betwee n beta-strand 3 and alpha-helix 3 of the (beta/alpha)(8)-barrel fold).