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.
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Kojima, K., Mikami-Sakaguchi, A., Kameya, M. et al. Substrate specificity engineering of Escherichia coli derived fructosamine 6-kinase. Biotechnol Lett 35, 253–258 (2013). https://doi.org/10.1007/s10529-012-1062-9
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DOI: https://doi.org/10.1007/s10529-012-1062-9