Cloned (Comment) | Organism |
---|---|
recombinant expression of GST-tagged wild-type and mutant enzymes in Escherichia coli strain JM109 | Neisseria polysaccharea |
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 | 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 | ? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Neisseria polysaccharea | Q9ZEU2 | - |
- |
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 | ? | - |
? | |
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 | ? | - |
? |
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 | Comment | Organism |
---|---|---|
NPAS | - |
Neisseria polysaccharea |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Neisseria polysaccharea |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7 | - |
assay at | Neisseria polysaccharea |
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 |