Substrate specificity engineering of Escherichia coli derived fructosamine 6-kinase.
Biotechnol Lett
; 35(2): 253-8, 2013 Feb.
Article
em En
| MEDLINE
| ID: mdl-23076362
ABSTRACT
A three-dimensional structural model of Escherichia coli fructosamine 6-kinase (FN6K), an enzyme that phosphorylates fructosamines at C6 and catalyzes the production of the fructosamine 6-phosphate stable intermediate, was generated using the crystal structure of 2-keto-3-deoxygluconate kinase isolated from Thermus thermophilus as template. The putative active site region was then investigated by site-directed mutagenesis to reveal several amino acid residues that likely play important roles in the enzyme reaction. Met220 was identified as a residue that plays a role in substrate recognition when compared to Bacillus subtilis derived FN6K, which shows different substrate specificity from the E. coli FN6K. Among the various Met220-substituted mutant enzymes, Met220Leu, which corresponded to the B. subtilis residue, resulted in an increased activity of fructosyl-valine and decreased activity of fructosyl-lysine, thus increasing the specificity for fructosyl-valine by 40-fold.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfotransferases
/
Engenharia de Proteínas
/
Frutosamina
/
Escherichia coli
/
Engenharia Metabólica
Idioma:
En
Revista:
Biotechnol Lett
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Japão