The x-ray structure of fructosamine 6-kinase (FN6K) derived from Escherichia coli (EcFN6K), which is expected as the sensing molecule for the glycated albumin (GA) measurement was determined in this study. Glycated proteins, such as hemoglobin A1c (HbA1c) or GA are the important long-term glycemic control markers for diabetes patient. FN6K catalyzes the phosphorylation of fructosyl amino acids using ATP and produces fructosyl amino acid 6-phosphate and ADP. Extensive studies in E. coli have led to an understanding of the physiological role of FN6K as the key enzyme in the catabolic pathway of naturally occurring fructosyl amino acids, which is supported by the reports that growth on Amadori products as sole carbon and nitrogen source is possible for E. coli. Fructosyl amino acid/peptide oxidase (FAOx/FPOx) are the current standard enzyme for the enzymatic glycated protein measurement. FN6K does not react with any sugars or free amino acids, and are thus specific for fructosyl amino acid. FN6Ks show lower Km values than FAOXs/FPOXs, while FN6K and FAOXs/FPOXs have comparable Vmax values. These properties together with its robustness in the recombinant production, FN6K will provide alternative detection principles and its utilization as novel molecular recognition elements for glycated protein sensing. In this study, x-ray structure of EcFN6K was determined. To confirm the roles of elucidated residues which are predicted to serve significant roles in catalytic reaction and substrate recogition, the mutational studies were also carried out. As the result, the x-ray structure of EcFN6K as the complex with ATP analog, Adenylyl-imidodiphosphate (AMPPNP) was obtained. The overall structure revealed that FN6K belongs to ribokinase family. Comparing the cavity for substrate entrance, that of EcFN6K was observed to be remarkably wider than those of FAOx/FPOxs. This observation suggested that the large substrate entrance and pocket in FN6K make it possible to react with large substrate such as intact GA. The mutational studies suggested that the conserved residues among in the other ribokinase family enzymes in FN6K are identified as the catalytic residue and residues recognizing the phosphate group of ATP.

X-ray structure of fructosamine 6-kinase, a future biorecognition molecule for glycated protein sensing for the glycemic control in diabetic patients

ACS Fall 2021

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