Crystallization and preliminary X-ray studies of recombinant amylosucrase from<i>Neisseria polysaccharea</i>

Skov, L.K.; Mirza, Osman; Henriksen, A.; Montalk, Gabrielle Potocki de; Remaud‐Siméon, Magali; Sarçabal, Patricia; Willemot, René‐Marc; Monsan, Pierre; Gajhede, Michael

doi:10.1107/s0907444999015887

Cited by 27 publications

(36 citation statements)

References 10 publications

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“…In CD2, the corresponding dipeptide NV is replaced with 2214 FI, which can also be found in the amylosucrase sequence. Structural data obtained for this enzyme (52) suggest that the Phe residue could be engaged in the specificity towards the fructo-furanosyl ring of sucrose (53). Moreover, the Ile residue at position 2215 is also found in GTF-A from L. reuteri (59) and is strongly conserved in the ␣-amylase family.…”

Section: Discussionmentioning

confidence: 97%

“…This led to a new classification of the glucansucrase enzymes in family 70 of the glycoside hydrolases on the CAZy website (http://afmb.cnrs-mrs.fr/ϳpedro/CAZY/db.html). The only exception is amylosucrase from Neisseria polysaccharea, which belongs to family 13 since its structure is not circularly permutated and is closely related to the ␣-amylase structure with a much lower molecular size than that of the glucansucrase enzymes (40,52).…”

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confidence: 99%

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Molecular Characterization of DSR-E, an α-1,2 Linkage-Synthesizing Dextransucrase with Two Catalytic Domains

et al. 2002

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A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of ␣-1,6 and ␣-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and ␣-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.Glucansucrase enzymes from oral streptococci, Leuconostoc mesenteroides strains, and some Lactobacillus and Neisseria sp. catalyze the transfer of glucosyl residues from sucrose to synthesize ␣-D-glucopyranosyl homopolymers and oligomers. When sucrose is the sole substrate, high-molecular-weight polymers are obtained. Depending on the glucansucrase-producing strain, the synthesized glucans differ in size and structure. When efficient acceptors, such as maltose or isomaltose, are added to the reaction medium, glucansucrases catalyze the synthesis of low-molecular-weight oligosaccharides and the regiospecificity of several dextransucrases (type of linkages) from the Leuconostoc genus is conserved in oligosaccharide synthesis (8,13,41,45).To date, 17 glucosyltransferase (GTF)-encoding genes from Streptococcus spp., 8 glucansucrase-encoding genes from L. mesenteroides, and 1 gene from Lactobacillus reuteri have been cloned (for reviews, see references 3, 16, 32, and 59). Sequence information shows that they are closely related and share a common structure. These genes code for large enzymes with an average molecular mass of 160,000 Da composed of two main domains: an N-terminal conserved catalytic core of about 900 amino acids and a C-terminal domain covering 300 to 400 residues thought to be responsible for glucan binding and constituted by a series of repeated units (32). In addition, biochemical studies revealed that glucansucrases share many mechanistic features with amylolytic enzymes (15). Structural homologies were later confirmed by amino acid sequence comparison with glucoside hydrolases from family 13 (20). In family 13, the catalytic domain is formed of eight ␤-sheets alternating with eight ␣-helices, conferring a (␤/␣) 8 barrel structure (55). Two structure predictions (9, 26) concluded that glucansucrase enzymes also possess a catalytic (␤/␣) 8 barrel domain. However, MacGregor et al. (26) observed that well-recognized sequence segments appear in a different order, which tends to show that the ␤/␣ barrel elements are circularly permutate...

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Section: Discussionmentioning

confidence: 97%

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confidence: 99%

Molecular Characterization of DSR-E, an α-1,2 Linkage-Synthesizing Dextransucrase with Two Catalytic Domains

et al. 2002

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“…For family GH13 enzymes, high-resolution three-dimensional structures are available, often in complex with substrates, inhibitors, or products (207). The three-dimensional structures of amylosucrase (also in complex with sucrose, oligosaccharides, and with a covalently bound glucopyranosyl moiety) resemble those of other proteins of family GH13 (80,115,181,183). Sequence alignments, mutagenesis studies, and three-dimensional structure analyses within family GH13 have resulted in identification of the amino acid residues involved in catalysis (204).…”

Section: Fig 2 Topology Diagrams Of Members Of ␣-Amylase Family Gh1mentioning

confidence: 99%

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

Hijum

Kralj

Ozimek

et al. 2006

Microbiol Mol Biol Rev

391

315

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SUMMARY Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and α-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with α-amylase enzymes (family GH13), with a predicted permuted (β/α)8 barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of α-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize β-fructan polymers with either β-(2→6) (inulin) or β-(2→1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed β-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either β-(2→6) or β-(2→1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.

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“…However, Neisseria polysaccharea amylosucrase (NpAS) is the only AS for which several structures, alone or in complex with sucrose substrate or products, are available to date (17)(18)(19)(20). The three-dimensional structure of NpAS is organized in five domains, namely A, B, C, N, and BЈ.…”

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Structural Investigation of the Thermostability and Product Specificity of Amylosucrase from the Bacterium Deinococcus geothermalis

Guerin

Barbe

Pizzut-Serin

et al. 2012

Journal of Biological Chemistry

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Background: Amylosucrases (AS) hold great potential for glycodiversification. Results: The first three-dimensional structure of AS from Deinococcus geothermalis solved here revealed an unusual dimer organization. Structures of complex of AS with turanose were also determined. Conclusion: Dimerization may contribute to thermostability. Turanose versus trehalulose formation is controlled by residues from subsite ϩ1. Significance: This study improves the comprehension of AS properties and provides new insight for AS design.

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Crystallization and preliminary X-ray studies of recombinant amylosucrase fromNeisseria polysaccharea

Cited by 27 publications

References 10 publications

Molecular Characterization of DSR-E, an α-1,2 Linkage-Synthesizing Dextransucrase with Two Catalytic Domains

Molecular Characterization of DSR-E, an α-1,2 Linkage-Synthesizing Dextransucrase with Two Catalytic Domains

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

Structural Investigation of the Thermostability and Product Specificity of Amylosucrase from the Bacterium Deinococcus geothermalis

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