An active-site mutation causes enhanced reactivity and altered regiospecificity of transglucosylation catalyzed by the Bacillus sp. SAM1606 alpha-glucosidase.

Bacillus sp. SAM1606 alpha-glucosidase catalyzes the transglucosylation of sucrose to produce three regioisomers of the glucosylsucroses, with theanderose (6-O(G)-glucosylsucrose) as the most abundant transfer product. To find the active-site amino acid residues which can affect the reactivity and regiospecificity of the glucosyl transfer, 16 mutants with amino acid substitutions near the active site were allowed to react with 1.75 M sucrose at 60 degrees C, pH 6.0, and the course of transglucosylation as well as the product specificity were analyzed. The sites of the amino acid substitutions were selected by comparing the conserved amino acid sequences located near the active site of the SAM1606 enzyme with those of the Bacillus oligo-1,6-glucosidases (O16G), which have very high amino acid sequence similarities near the active site but have a distinct substrate specificity. The results showed that, among the mutated SAM1606 enzymes examined, only the mutants with substitution of Gly273 with Pro showed an altered reactivity and specificity of transglucosylation; these mutants exhibited a significantly enhanced initial velocity of glucosyl transfer, yielding isomelezitose (6-O(F)-glucosylsucrose) instead of theanderose as the major transfer product. These results indicate that the substitution of Gly273 with Pro critically governs the enhanced reactivity and altered specificity of the transglucosylation. The notion that the amino acid residue at this position is the determinant of the glucosyl-transfer specificity was further confirmed by observation that the Bacillus cereus O16G, which has a proline at the corresponding position, produced isomelezitose as the major transfer product during transglucosylation with sucrose.

Mutational analysis of the role of His452 of Saccharopolyspora rectivirgula beta-galactosidase.

To examine the role of His452 of the Saccharopolyspora rectivirgula beta-galactosidase in the binding of a tightly bound, catalytically important Mn2+ (i.e., class II Mn2+) ion, His452 was replaced with Phe or Glu and the respective site-directed mutants, H452F and H452E, were characterized. Neither mutant contained Mn2+ in an Mn2+-free buffer and both were virtually inactive in the absence of Mn2+ (their relative activities being less than 0.03% that of the fully activated wild-type enzyme). When Mn2+ was added, however, the mutants were activated to 3% (for H452F) and 0.8% (for H452E) of the full activity of the wild type. The Mn2+ concentrations needed for half-maximal activation of H452F and H452E were, respectively, 15,000 and 5000 times higher than the reported dissociation constant (2 nM) of the class II Mn2+, suggesting that His452 plays a key role in the binding of this catalytically important Mn2+. Activation of the mutants by Mn2+, albeit very weak, contrasts with a lack of any such metal activation previously observed with the two corresponding mutants of Escherichia coli lacZ beta-galactosidase.

Unique primary structure of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula.

The gene of the monomeric multimetal beta-galactosidase of Saccharopolyspora rectivirgula was cloned and sequenced. Although the enzyme could be assigned as a member of beta-galactosidases belonging to the glycosyl hydrolase family 2, it has unusual structural features for beta-galactosidase of this family; it contained a unique sequence which consists of approximately 200 amino acid residues with no similarity to known proteins. This 200-residue sequence exists as if it is inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme.

Altering substrate specificity of Bacillus sp. SAM1606 alpha-glucosidase by comparative site-specific mutagenesis.

The Bacillus sp. SAM1606 alpha-glucosidase with a broad substrate specificity is the only known alpha-glucosidase that can hydrolyze alpha,alpha'-trehalose efficiently. The enzyme exhibits a very high sequence similarity to the oligo-1,6-glucosidases (O16G) of Bacillus thermoglucosidasius and Bacillus cereus which cannot act on trehalose. These three enzymes share 80% identical residues within the conserved regions (CR), which have been suggested to be located near or at the active site of the alpha-amylase family enzymes. To identify by site-specific mutagenesis the critical residues that determine the broad substrate specificity of the SAM1606 enzyme we compared the CR sequences of these three glucosidases and selected five targets to be mutagenized in SAM1606 alpha-glucosidase, Met76, Arg81, Ala116, Gly273, and Thr342. These residues have been specifically replaced by in vitro mutagenesis with Asn, Ser, Val, Pro, and Asn, respectively, as in the Bacillus O16G. The 12 mutant enzymes with single and multiple substitutions were expressed and characterized kinetically. The results showed that the 5-fold mutation virtually abolished the affinity of the enzyme for alpha, alpha'-trehalose, whereas the specificity constant for the hydrolysis of isomaltose, a good substrate for both the SAM1606 enzyme and O16G, remained essentially unchanged upon the mutation. This loss in affinity for trehalose was critically governed by a Gly273 --> Pro substitution, whose effect was specifically enhanced by the Thr342 --> Asn substitution in the 5-fold and quadruple mutants. These results provide evidence for the differential roles of the amino acid residues in the CR in determining the substrate specificity of the alpha-glucosidase.