ALTERING SUBSTRATE-SPECIFICITY OF BACILLUS SP SAM1606 ALPHA-GLUCOSIDASE BY COMPARATIVE SITE-SPECIFIC MUTAGENESIS

The Bacillus sp. SAM1606 alpha-glucosidase with a broad substrate spec ificity is the only known alpha-glucosidase that can hydrolyze alpha,a lpha'-trehalose efficiently. The enzyme exhibits a very high sequence similarity to the oligo 1,6-glucosidases (O16G) of Bacillus thermogluc osidasius and Bacillus cereus which cannot act on trehalose. These thr ee enzymes share 80% identical residues within the conserved regions ( CR), which have been suggested to be located near or at the active sit e of the alpha-amylase family enzymes. To identify by site-specific mu tagenesis the critical residues that determine the broad substrate spe cificity of the SAM1606 enzyme we compared the CR sequences of these t hree glucosidases and selected five targets to be mutagenized in SAM16 06 alpha-glucosidase, Met(76), Arg(81), Ala(116), Gly(273), and Thr(34 2) These residues have been specifically replaced by in vitro mutagene sis with Asn, Ser, Val, Pro, and Asn, respectively, as in the Bacillus O16G. The 12 mutant enzymes with single and multiple substitutions we re expressed and characterized kinetically. The results showed that th e 5-fold mutation virtually abolished the affinity of the enzyme for a lpha,alpha'-trehalose, whereas the specificity constant for the hydrol ysis of isomaltose, a good substrate for both the SAM1606 enzyme and O 16G, remained essentially unchanged upon the mutation. This loss in af finity for trehalose was critically governed by a Gly(273) --> Pro sub stitution, whose effect was specifically enhanced by the Thr(342) --> Asn substitution in the B-fold and quadruple mutants. These results pr ovide evidence for the differential roles of the amino acid residues i n the CR in determining the substrate specificity of the alpha-glucosi dase.