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Literature summary for 2.4.1.4 extracted from

  • Daude, D.; Topham, C.M.; Remaud-Simeon, M.; Andre, I.
    Probing impact of active site residue mutations on stability and activity of Neisseria polysaccharea amylosucrase (2013), Protein Sci., 22, 1754-1765.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of GST-tagged wild-type and mutant enzymes in Escherichia coli strain JM109 Neisseria polysaccharea

Protein Variants

Protein Variants Comment Organism
D144A site-directed mutagenesis Neisseria polysaccharea
D144E site-directed mutagenesis Neisseria polysaccharea
D144I site-directed mutagenesis Neisseria polysaccharea
D507A site-directed mutagenesis Neisseria polysaccharea
D507I site-directed mutagenesis Neisseria polysaccharea
E328Q inactive mutant, sucrose binding structure analysis using the crystal structure, PDB ID 1JGI Neisseria polysaccharea
F250A site-directed mutagenesis Neisseria polysaccharea
F250N site-directed mutagenesis Neisseria polysaccharea
F250Y site-directed mutagenesis Neisseria polysaccharea
H187L site-directed mutagenesis Neisseria polysaccharea
H187Q site-directed mutagenesis Neisseria polysaccharea
H392P site-directed mutagenesis Neisseria polysaccharea
additional information introduction of mutations at D144, Y147, F250, R284, and R509 positions leads to equivalent or impaired stability compared with the wild-type enzyme. Several mutant variants retain their transglucosylation activity and are still able to catalyze the synthesis of maltooligosaccharides. In particular, two mutants H392P and Y147F display original and controlled product distributions compared to the wild-type parental NpAS being more efficient for synthesizing soluble oligosaccharides. Two H187 variants and nine H392 variants show lower free energy values than that calculated for the wild-type enzyme Neisseria polysaccharea
R284D site-directed mutagenesis Neisseria polysaccharea
R284H site-directed mutagenesis Neisseria polysaccharea
R284K site-directed mutagenesis Neisseria polysaccharea
R284V site-directed mutagenesis Neisseria polysaccharea
R446A site-directed mutagenesis Neisseria polysaccharea
R446E site-directed mutagenesis Neisseria polysaccharea
R446F site-directed mutagenesis Neisseria polysaccharea
R509Q site-directed mutagenesis Neisseria polysaccharea
Y147A site-directed mutagenesis Neisseria polysaccharea
Y147F site-directed mutagenesis Neisseria polysaccharea
Y147N site-directed mutagenesis Neisseria polysaccharea

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
additional information Neisseria polysaccharea the amylosucrase from Neisseria polysaccharea naturally catalyzes the synthesis of alpha-glucans from the widely available donor sucrose. NpAS is highly specific for its natural substrate and subsite -1 (according to GH nomenclature) plays a major role in the recognition of the sucrose glucosyl moiety through a highly efficient hydrogen bonding interaction network, subsite 21 is responsible for the high affinity for sucrose ?
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Organism

Organism UniProt Comment Textmining
Neisseria polysaccharea Q9ZEU2
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Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information the amylosucrase from Neisseria polysaccharea naturally catalyzes the synthesis of alpha-glucans from the widely available donor sucrose. NpAS is highly specific for its natural substrate and subsite -1 (according to GH nomenclature) plays a major role in the recognition of the sucrose glucosyl moiety through a highly efficient hydrogen bonding interaction network, subsite 21 is responsible for the high affinity for sucrose Neisseria polysaccharea ?
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additional information hydrolysis of p-nitrophenyl-alpha-D-glucopyranoside is used for activity measurements. Substrate specificities of recombinant wild-type and mutant enzymes, overview Neisseria polysaccharea ?
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Subunits

Subunits Comment Organism
More the residues forming subsites -1 and +1 are considered to be likely involved in the activity as well as the overall stability of the enzyme, active site structure, overview Neisseria polysaccharea

Synonyms

Synonyms Comment Organism
NPAS
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Neisseria polysaccharea

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
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assay at Neisseria polysaccharea

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
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assay at Neisseria polysaccharea

General Information

General Information Comment Organism
evolution amylosucrase from Neisseria polysaccharea is a transglucosidase from the GH13 family of glycoside-hydrolases. Natural molecular evolution has modeled a dense hydrogen bond network at subsite 21 responsible for the specific recognition of sucrose and conversely, it has loosened interactions at the subsite 11 creating a highly promiscuous subsite 11. The residues forming these subsites are considered to be likely involved in the activity as well as the overall stability of the enzyme Neisseria polysaccharea
additional information essential role of enzyme subsite -1 for protein fitness and robustness Neisseria polysaccharea