Sucrose 6-.alpha.-D-glucosyltransferase from Streptococcus sobrinus: characterization of a glucosyl-enzyme complex

Abstract

A covalent glucosyl-enzyme was isolated from a quenched reaction of Streptococcus sobrinus sucrose 6-alpha-D-glucosyltransferase and radiolabeled sucrose. No complex was observed with heat-inactivated enzyme or when sucrose was replaced with radiolabeled maltose or glucose. The complex was stable at pH 2 in 1% sodium dodecyl sulfate, 6.0 M urea, and 4.0 M guanidine hydrochloride, but became increasingly labile with increased pH (32-min half-life at pH 7.0). D-Glucose was the exclusive radiolabeled compound identified when all radioactivity was released under mild alkaline conditions. Glucosyl-enzyme hydrolysis rates were linearly dependent on hydroxide ion concentration, giving a second-order rate constant of 2.15 x 10(5) M-1 min-1. When compared to the base lability of known glycosyl amino acid derivatives, the pH dependency of the glucosyl-enzyme most closely paralleled a glucosyl linkage to a carboxyl group. A novel application of a carbohydrate high-performance liquid chromatography column in aqueous solution was used to identify the anomeric form of D-glucose released on (i) alkaline hydrolysis of denatured glucosyl-enzyme and (ii) native enzyme hydrolysis of sucrose. The beta-anomer was identified in the former case and the alpha-anomer in the latter. The results with the denatured glucosyl-enzyme are consistent with a beta-glucosyl ester linkage to an aspartic or glutamic acid that hydrolyzes at the ester carbon with retention of anomeric configuration; for native glucosyltransferase catalysis, the data are consistent with a beta-glucosyl covalent intermediate as well, where deglucosylation occurs by attack at the acetal carbon with anomeric inversion.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

“…1D) is involved in the formation of the covalent glucosyl-enzyme complex (80,111,128,129,204). Its mutagenesis in family GH13 and family GH70 enzymes resulted in drastic loss of enzyme activity (38,83,88,99,119,165).…”

A circularly permuted α‐amylase‐type α/β‐barrel structure in glucan‐synthesizing glucosyltransferases

“…There is evidence that for ~-amylases a covalent intermediate (II) may form between the glycon part of the substrate and a carboxyl group on the enzyme [51]. For glucosyl transferases strong evidence supports the occurrence of a glucosyl-glucosyltransferase intermediate like II [1,4,11,52,53]. The esterified carboxyl group of II has been identified as that of Asp 415 of GTF-I or Asp 437 of GTF-S [11].…”

Effect of carbohydrate fatty acid esters on Streptococcus sobrinus and glucosyltransferase activity

“…Mutans streptococci secrete at least three glucosyltransferases: two synthesize a-(1 fi 6)-linked water-soluble glucans (dextrans), which differ in glucan affinity and degree of a-(1 fi 3)-branching, and a third synthesizes an a-(1 fi 3)-linked water-insoluble glucan (mutan). 3 GTFs belong to the family of glycansucrases (glucansucrases and fructansucrases), bacterial enzymes acting on sucrose and utilizing it as the sole energy source for sugar synthesis. 4 All glycansucrases share the same substrate specificity, but catalyze the synthesis of a variety of oligo-and polysaccharides.…”