Identification of an archaeal maltooligosyltrehalose trehalohydrolase encoded by an interrupted gene.

Complete genome analysis of the thermoacidophilic Archaeon Sulfolobus tokodaii strain 7 revealed two open reading frames (ORF), namely, ST0926 and ST0927. These ORFs are interrupted by two putative insertions and encode for the N- and C-terminal fragments, respectively, of a putative Sulfolobus sp. maltooligosyltrehalose trehalohydrolase (StMTHase). Two specific deletion mutations, designed on the basis of sequence alignments of the adjacent ORFs and the published Sulfolobus sp. MTHases, allowed soluble expression in Escherichia coli of an active acidic and thermophilic enzyme. The purified enzyme exhibited a maximum amylolytic activity at 70 °C and pH 5.0, which resembled the optimal conditions of the Sulfolobus homologs. Furthermore, we report that these ORFs are actively co-transcribed in vivo, and we confirm the presence of insertions between them at the cDNA level. However, immunization and western blot experiments demonstrated no expression of ST0926 or the putative full-length StMTHase in vivo, indicating that they might exist as nonfunctional pseudogenes.