Cloning and Characterization of Fructosamine-6-Kinase from Arthrobacter aurescens

Abstract

Fructosamine-6-kinases (FN6Ks) that catalyze phosphorylation of glycated amino acids, i.e., fructosyl amino acids (FAs), have been shown as a potential recognition element for glycated protein detection. However, there are only two available FN6Ks: those from Escherichia coli which is specific for ε-fructosyl lysine (ε-FK) and Bacillus subtilis which recognizes both ε-FK and α-FA as substrates. In this study, we characterized an FN6K homologue isolated from Arthrobacter, some of whose species are reported to assimilate FA. The BLAST searches of Arthrobacter genomic database, using the bacterial FN6K primary structure information, revealed the presence of an FN6K homologue in Arthrobacter aurescens TC1 strain. Indeed, enzymatic assays confirmed that the putative FN6K from A. aurescens is an FN6K that is specific for ε-FK, although the primary sequence alignments showed similarity of A. aurescens FN6Ks with FN6Ks from B. subtilis and E. coli at the same level. In this study, we describe for the first time the presence of FN6K in Arthrobacter spp. and ε-FK-specific degradation pathway from Gram-positive bacteria, providing important information for the development of FA-recognizing molecules as well as for the FA assimilation system in bacteria.

Advancing the Development of Glycated Protein Biosensing Technology

“…FN6Ks have been identified in Escherichia coli, Bacillus subtilis, and Arthrobacter aurescens. [22][23][24] In these bacteria, FN6K catalyzes the production of fructosamine 6-phosphate by phosphorylating fructosamine at the C6 of the deoxyfructose moiety. Following phosphorylation, deglycase catalyzes the degradation of fructosamine 6-phosphate to free amino acid and glucose 6-phosphate ( Figure 1C).…”

“…22,36 The substrate specificity of the B. subtilis FN6K differs from those of the E. coli and A. aurescens FN6Ks, although they share 50% similarity in amino acid sequences. 24 The FN6K from B. subtilis shows broader substrate specificity, catalyzing the phosphorylation of both ε-FK and α-glycated amino acids, such as fructosyl valine. In contrast, the FN6Ks from E. coli and A. aurescens preferentially catalyze the phosphorylation of ε-FK (Table 1).…”

“…Kinases and binding proteins that recognize glycated amino acids and glycated proteins have also recently been reported. [21][22][23][24][25][26] These proteins have been reported to have low K m values for their substrates and the ability to recognize intact glycated proteins, giving them great potential as novel biosensing molecules. Engineering of these proteins is expected to expand the development of more sensitive and simple biosensing systems for glycated proteins as alternatives to the FAOX/FPOX-based systems.…”

Electrochemical sensing system employing fructosamine 6‐kinase enables glycated albumin measurement requiring no proteolytic digestion

“…FN6K catalyzes the production of fructosamine 6‐phosphate by phosphorylating fructosamine at C6 of the deoxyfructose moiety, which is followed by the degradation of fructosamine 6‐phosphate to the free amino acid and glucose 6‐phosphate, catalyzed by deglycase . FN6Ks have been identified in Escherichia coli and Bacillus subtilis , and our group has reported the FN6K from Arthrobacter aurescens . The FN6Ks from E. coli and A. aurescens preferentially catalyze the phosphorylation of ε‐fructosyl lysine (ε‐FK), while the FN6K from B. subtilis shows broader substrate specificity against both ε‐FK and α‐glycated amino acids.…”

“…We previously reported that the enzymatic activity of FN6Ks for ε‐FK can be measured colorimetrically by coupling with pyruvate kinase, pyruvate oxidase, and peroxidase reactions and detecting quinoneimine dye, as shown in Fig. . The application of this sensing scheme for the detection of GA is expected to yield a novel sensing system which is rapid, reproducible, and suitable for automated analyzers.…”