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

MacGregor, E. Ann; Jespersen, Hans M.; Svensson, Birte

doi:10.1016/0014-5793(95)01428-4

Cited by 147 publications

(201 citation statements)

References 59 publications

(106 reference statements)

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“…Unlike glucansucrases of GH family 70 from lactic acid bacteria (dextransucrase, alternansucrase, and mutansucrase) (17), for which a circularly permutated (␤/␣) 8 -barrel-fold is predicted (18), the catalytic A-domain of AS adopts a nonpermutated (␤/␣) 8 -barrel fold like all of the enzymes of GH family 13 (19). Structural analyses supported by single-site mutational experiments enabled the assignment of the nucleophile (Asp 286 ) and of the acid/base catalyst (Glu 328 ) (20).…”

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confidence: 99%

Molecular Basis of the Amylose-like Polymer Formation Catalyzed by Neisseria polysaccharea Amylosucrase

Albenne

Skov

Mirza

et al. 2004

Journal of Biological Chemistry

102

114

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Amylosucrase from Neisseria polysaccharea is a remarkable transglucosidase from family 13 of the glycoside-hydrolases that synthesizes an insoluble amyloselike polymer from sucrose in the absence of any primer. Amylosucrase shares strong structural similarities with ␣-amylases. Exactly how this enzyme catalyzes the formation of ␣-1,4-glucan and which structural features are involved in this unique functionality existing in family 13 are important questions still not fully answered. Here, we provide evidence that amylosucrase initializes polymer formation by releasing, through sucrose hydrolysis, a glucose molecule that is subsequently used as the first acceptor molecule. Maltooligosaccharides of increasing size were produced and successively elongated at their nonreducing ends until they reached a critical size and concentration, causing precipitation. The ability of amylosucrase to bind and to elongate maltooligosaccharides is notably due to the presence of key residues at the OB1 acceptor binding site that contribute strongly to the guidance (Arg 415 , subsite ؉4) and the correct positioning (Asp 394 and Arg 446 , subsite ؉1) of acceptor molecules. On the other hand, Arg 226 (subsites ؉2/؉3) limits the binding of maltooligosaccharides, resulting in the accumulation of small products (G to G3) in the medium. A remarkable mutant (R226A), activated by the products it forms, was generated. It yields twice as much insoluble glucan as the wild-type enzyme and leads to the production of lower quantities of by-products.Amylosucrase (EC 2.4.1.4) is a glucansucrase belonging to glycoside-hydrolase (GH) 1 family 13 (1, 2). 2 This transglucosidase catalyzes the synthesis of an insoluble amylose-like polymer from sucrose (3), a cheap and easily available agroresource. This is in contrast to starch or glycogen synthases (4), which require nucleotide-activated sugar as a donor. Amylosucrase is thus attractive for the industrial synthesis of amyloselike polymers and for the modification of glucans (in particular to form nondigestible glucans) (5). Remarkably, amylosucrase is the only member of GH family 13 displaying polymerase activity and is clearly unique in this family that mainly contains starch-degrading enzymes. Amylosucrase was first isolated in the culture supernatant of Neisseria perflava (3) and later identified in various Neisseria strains (6, 7). Recently, data mining has revealed the presence of genes encoding putative amylosucrases in the genome of many other organisms such as Deinococcus radiodurans (8), Caulobacter crescentus (9), Xanthomonas campestris, Xanthomonas axonopodis (10), and Pirellula sp. (11). Recombinant amylosucrase from Neisseria polysaccharea (AS) has been the most extensively studied amylosucrase. The gene encoding AS (1) has been cloned, and its product has been purified to homogeneity. Characterization of the reaction products synthesized from sucrose substrate showed that sucrose isomers (turanose and trehalulose), glucose, maltose, and maltotriose were also produced besides the insoluble...

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confidence: 99%

Molecular Basis of the Amylose-like Polymer Formation Catalyzed by Neisseria polysaccharea Amylosucrase

Albenne

Skov

Mirza

et al. 2004

Journal of Biological Chemistry

102

114

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“…The members of the GTF family are highly similar and the enzyme that we have chosen is a representative of the group. Recently, there has been an alternative alignment based on circular permutation by MacGregor et al ( 1996). …”

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confidence: 99%

Knowledge‐based model of a glucosyltransferase from the oral bacterial group of mutans streptococci

et al. 1997

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Mutans streptococci glucosyltransferases catalyze glucosyl transfer from sucrose to a glucan chain. We previously identified an aspartyl residue that participates in stabilizing the glucosyl transition state. The sequence surrounding the aspartate was found to have substantial sequence similarity with members of a-amylase family. Because little is known of the protein structure beyond the amino acid sequence, we used a knowledge-based interactive algorithm, MACAW, which provided significant level of homology with a-amylases and glucosyltransferase from Streptococcus downei gtfl (GTF). The significance of GTF similarity is underlined by GTF/a-amylase residues conserved in all but one a-amylase invariant residues. Site-directed mutagenesis of the three GTF catalytic residues are homologous with the a-amylase catalytic triad. The glucosyltransferases are members of the 4/7-superfamily that have a (P/a)8-barrel structure and belong to family 13 of the glycohydralases.Keywords: (P/a)x-barrel; catalytic residues; enzymes; mutans streptococci glucosyltransferase; protein modeling; site-directed mutagenesis Oral bacteria referred to as mutans streptococci secrete glucosyltransferases (EC2.4.1.5), which are a significant virulence factor in initiation of dental caries on smooth enamel surfaces and cementa1 root surfaces (Hamada & Slade, 1980;Loesche, 1986). The extracellular enzymes catalyze glucosyl transfer from sucrose to a growing glucan chain. The bacterial colony produces metabolic acids that demineralize the tooth surface and, if unchecked, will lead to dental caries.GTFs are very large proteins of approximately 1,300-1,700 residues. The enzyme can be grossly divided into an N-terminal catalytic domain and a C-terminal glucan-binding domain that captures the synthesized polysaccharide (Ferretti et al., Wong et al., 1990). The large size of the enzyme has made it difficult to develop structure information beyond the solution of the amino acid sequence. Nonetheless, we became aware of sequence similarity between GTF and members of the a-amylase family. In our earlier studies, we trapped a glucosyl-enzyme catalytic intermediate (Mooser & Iwaoka, 1989) that was coordinated with an aspartyl Reprint requests to: Gregory Mooser, Department of Basic Sciences, School of Dentistry, University of Southern California, Los Angeles, California 90089-0641; e-mail: gmooser@hsc.usc.edu.Abbreviations: GTF, glucosyltransferase from Streptococcus downei g@; MACAW, multiple alignment construction and analysis workbench; SP, sum of the pairs; dNJ, I-deoxynojirimycin. residue, and the sequence surrounding the aspartate was homologous with a sequence conserved in all members of the a-amylase family and several related enzymes (Mooser et al., Mooser, 1992).In this study, we coupled knowledge-based multiple sequence alignment with site-directed mutagenesis to establish evidence of structural similarity between GTF and solved crystal structures of members of the a-amylase family. The members of the GTF family are highly similar and ...

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“…evolution. Of the more than 100 GH families defined at present in CAZy, the typical starch hydrolases and related enzymes are found in families 13,14,15,31,57, 70 and 77 [3][4][5]. Family GH13 is well known as the a-amylase family and, together with GH70 and GH77, forms clan GH-H [6][7][8][9][10].…”

Section: Introductionmentioning

confidence: 99%

“…Family GH77 enzymes contain this (b/a) 8 -barrel, but lack domain C found C-terminal to the barrel in GH13 enzymes (see, for example [13]). Family GH70, in contrast, is believed to possess a circularly permuted version of the GH13-type (b/a) 8 -barrel [14]. Enzymes of the entire clan GH-H are characterised by several (from 4 to 7) conserved sequence regions [15,16] and a common catalytic machinery involving an aspartate in strand b4, a glutamate in strand b5, and an aspartate after strand b7 that are essential for activity [3][4][5][6][7][8][9][10][11]17].…”

Section: Introductionmentioning

confidence: 99%

A remote but significant sequence homology between glycoside hydrolase clan GH‐H and family GH31

2007

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Although both the a-amylase super-family, i.e. the glycoside hydrolase (GH) clan GH-H (the GH families 13, 70 and 77), and family GH31 share some characteristics, their different catalytic machinery prevents classification of GH31 in clan GH-H. A significant but remote evolutionary relatedness is, however, proposed for clan GH-H with GH31. A sequence alignment, based on the idea that residues equivalent in the primordial catalytic GH-H/GH31 (b/a) 8 -barrel may not be found in the present-day GH-H and GH31 structures at strictly equivalent positions, shows remote sequence homologies covering b3, b4, b7 and b8 of the GH-H and GH31 (b/a) 8 -barrels. Structure comparison of GH13 a-amylase and GH31 a-xylosidase guided alignment of GH-H and GH31 members for construction of evolutionary trees. The closest sequence relationship displayed by GH31 is to GH77 of clan GH-H.

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

Cited by 147 publications

References 59 publications

Molecular Basis of the Amylose-like Polymer Formation Catalyzed by Neisseria polysaccharea Amylosucrase

Molecular Basis of the Amylose-like Polymer Formation Catalyzed by Neisseria polysaccharea Amylosucrase

Knowledge‐based model of a glucosyltransferase from the oral bacterial group of mutans streptococci

A remote but significant sequence homology between glycoside hydrolase clan GH‐H and family GH31

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