SEQUENCING OF THE AMYLOPULLULANASE (APU) GENE OF THERMOANAEROBACTER-ETHANOLICUS 39E, AND IDENTIFICATION OF THE ACTIVE-SITE BY SITE-DIRECTEDMUTAGENESIS

The complete nucleotide sequence of the gene encoding the dual active amylopullulanase of Thermoanaerobacter ethanolicus 39E (formerly Clost ridium thermohydrosulfuricum) was determined. The structural gene (apu ) contained a single open reading frame 4443 base pairs in length, cor responding to 1481 amino acids, with an estimated molecular weight of 162,780. Analysis of the deduced sequence of apu with sequences of alp ha-amylases and alpha-1,6 debranching enzymes enabled the identificati on of four conserved regions putatively involved in substrate binding and in catalysis. The conserved regions were localized within a 2.9-ki lobase pair gene fragment, which encoded a M(r) 100,000 protein that m aintained the dual activities and thermostability of the native enzyme . The catalytic residues of amylopullulanase were tentatively identifi ed by using hydrophobic cluster analysis for comparison of amino acid sequences of amylopullulanase and other amylolytic enzymes. Asp597, Gl u626, and Asp703 were individually modified to their respective amide form, or the alternate acid form, and in all cases both alpha-amylase and pullulanase activities were lost, suggesting the possible involvem ent of 3 residues in a catalytic triad, and the presence of a putative single catalytic site within the enzyme. These findings substantiate amylopullulanase as a new type of amylosaccharidase.