Substrate specificity engineering of Escherichia coli derived fructosamine 6-kinase.
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.
Ultizyme International Ltd, 1-13-16, Minami, Meguro, Tokyo, Japan.
Katsuhiro
K
Kojima
Akane
A
Mikami-Sakaguchi
Miho
M
Kameya
Yusuke
Y
Miyamoto
Stefano
S
Ferri
Wakako
W
Tsugawa
Koji
K
Sode
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D019270:Fructosamine / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D060847:Metabolic Engineering; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering; D013379:Substrate Specificity; D014633:Valine / Q000031:analogs & derivatives / Q000378:metabolism
D050505:Mutant Proteins; C045420:fructosylvaline; C033186:fructosyl-lysine; D019270:Fructosamine; D010770:Phosphotransferases; D014633:Valine; D008239:Lysine