ABSTRACT
Amylosucrase (E.C. 2.4.1.4) is a member of Family 13 of the glycoside hydrolases (the alpha-amylases), although its biological function is the synthesis of amylose-like polymers from sucrose. The structure of amylosucrase from Neisseria polysaccharea is divided into five domains: an all helical N-terminal domain that is not similar to any known fold, a (beta/alpha)(8)-barrel A-domain, B- and B'-domains displaying alpha/beta-structure, and a C-terminal eight-stranded beta-sheet domain. In contrast to other Family 13 hydrolases that have the active site in the bottom of a large cleft, the active site of amylosucrase is at the bottom of a pocket at the molecular surface. A substrate binding site resembling the amylase 2 subsite is not found in amylosucrase. The site is blocked by a salt bridge between residues in the second and eight loops of the (beta/alpha)(8)-barrel. The result is an exo-acting enzyme. Loop 7 in the amylosucrase barrel is prolonged compared with the loop structure found in other hydrolases, and this insertion (forming domain B') is suggested to be important for the polymer synthase activity of the enzyme. The topology of the B'-domain creates an active site entrance with several ravines in the molecular surface that could be used specifically by the substrates/products (sucrose, glucan polymer, and fructose) that have to get in and out of the active site pocket.
Subject(s)
Glucosyltransferases/metabolism , alpha-Amylases/metabolism , Amino Acid Sequence , Binding Sites , Glucosyltransferases/chemistry , Models, Chemical , Models, Molecular , Molecular Sequence Data , Protein FoldingABSTRACT
Amylosucrase from Neisseria polysaccharea catalyzes the synthesis of an amylose-like polymer from sucrose. Sequence alignment revealed that it belongs to the glycoside hydrolase family 13. Site-directed mutagenesis enabled the identification of functionally important amino acid residues located at the active center. Asp-294 is proposed to act as the catalytic nucleophile and Glu-336 as general acid base catalyst in amylosucrase. The conserved Asp-401, His-195 and His-400 residues are critical for the enzymatic activity. These results provide strong support for the predicted close structural and functional relationship between the sucrose-glucosyltransferases and enzymes of the alpha-amylase family.
Subject(s)
Amino Acids/analysis , Glucosyltransferases/chemistry , Neisseria/enzymology , Amino Acid Sequence , Aspartic Acid , Base Sequence , Binding Sites , Catalysis , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Glutamic Acid , Histidine , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Alignment , Structure-Activity RelationshipABSTRACT
Amylosucrase is a glucosyltransferase that synthesises an insoluble alpha-glucan from sucrose. The catalytic properties of the highly purified amylosucrase from Neisseria polysaccharea were characterised. Contrary to previously published results, it was demonstrated that in the presence of sucrose alone, several reactions are catalysed, in addition to polymer synthesis: sucrose hydrolysis, maltose and maltotriose synthesis by successive transfers of the glucosyl moiety of sucrose onto the released glucose, and finally turanose and trehalulose synthesis - these two sucrose isomers being obtained by glucosyl transfer onto fructose. The effect of initial sucrose concentration on initial activity demonstrated a non-Michaelian profile never previously described.
Subject(s)
Glucosyltransferases/metabolism , Neisseria/enzymology , Sucrose/metabolism , Catalysis/drug effects , Chromatography, High Pressure Liquid , Disaccharides/metabolism , Dose-Response Relationship, Drug , Fructose/metabolism , Fructose/pharmacology , Glucose/metabolism , Glucosyltransferases/isolation & purification , Hydrolysis/drug effects , Isomerism , Kinetics , Magnetic Resonance Spectroscopy , Maltose/metabolism , Polymers/chemistry , Polymers/metabolism , Solubility , Sucrose/chemistry , Sucrose/pharmacology , Trisaccharides/metabolismABSTRACT
Recombinant amylosucrase from Neisseria polysaccharea was crystallized by the vapour-diffusion procedure in the presence of polyethylene glycol 6000. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 95.7, b = 117.2, c = 62.1 A, and diffract to 1.6 A resolution. A p-chloromercuribenzene sulfonate (pcmbs) derivative has been identified and a selenomethionine-substituted protein has been produced and crystallized.
Subject(s)
Glucosyltransferases/chemistry , Neisseria/enzymology , Recombinant Proteins/chemistry , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Circular Dichroism , Crystallization , Crystallography, X-Ray , Escherichia coli/chemistry , Escherichia coli/enzymology , Glucosyltransferases/biosynthesis , Glucosyltransferases/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purificationABSTRACT
The gene encoding alternansucrase (ASR) from Leuconostoc mesenteroides NRRL B-1355, an original sucrose glucosyltransferase (GTF) specific to alternating alpha-1,3 and alpha-1,6 glucosidic bond synthesis, was cloned, sequenced and expressed into Escherichia coli. Recombinant enzyme catalyzed oligoalternan synthesis from sucrose and maltose acceptor. From sequence comparison, it appears that ASR possesses the same domains as those described for GTFs specific to either contiguous alpha-1,3 osidic bond or contiguous alpha-1,6 osidic bond synthesis. However, the variable region and the glucan binding domain are longer than in other GTFs (by 100 and 200 amino acids respectively). The N-catalytic domain which presents 49% identity with the other GTFs from L. mesenteroides possesses the three determinants potentially involved in the glucosyl enzyme formation.
