Substrate specificity engineering of Escherichia coli derived fructosamine 6-kinase

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.

Advancing the Development of Glycated Protein Biosensing Technology

“…Substitution of Met220 to Leu, the residue at the corresponding position in the B. subtilis FN6K, which shows a different substrate specificity, resulted in 7-fold Calibration curve for ε-fructosyl lysine by colorimetric assay (n = 2, error bar: SD). 36 Measurement of absorbance at 546 nm was carried out with fructosamine 6-kinase (FN6K) from E. coli and various concentrations of ε-fructosyl lysine (ε-FK), prepared by incubating glucose and lysine, in the reaction mixture (1 mM ATP, 1 mM MgCl 2 , 500 U/ml pyruvate kinase, 100 U/ml pyruvate oxidase, 1.5 mM N,N-bis(4-sulfobutyl)-3-methylaniline, 2 U/ml peroxidase, 1.5 mM 4-aminoantipyrine, 25 mM HEPES buffer [pH 7.1]) at 25°C. 36 The x-axis represents final concentrations of ε-FK in the reaction mixture.…”

Characterization of a Salmonella sugar kinase essential for the utilization of fructose-asparagine

“…Here, we demonstrate that FraD, the second enzyme in this pathway, indeed transforms F-Asp to 6-P-F- Because the functional roles for the structural genes in the fra locus were predicted based on the E. coli frl operon that permits utilization of F-Lys (Wiame et al 2002), it is instructive to compare the kinetic parameters of Salmonella FraD and E. coli FrlD, which converts F-Lys to 6-P-F-Lys. FrlD exhibits a k cat /K M = 45,000 min -1 mM -1 (Wiame et al 2004; also, see Kojima et al 2013), nearly five-fold greater than the 8265 min -1 mM -1 that we determined for FraD ( Fig. 3).…”

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

“…The gene encoding FN6K ( frlD ) was amplified and subcloned into a modified pET‐30c vector (Invitrogen) containing a 6‐Histidine‐tag as previously described . E. coli BL21 (DE3) cells were transfected with the constructed vector and grown aerobically at 37°C in LB medium containing kanamycin at 40 μg/mL.…”

“…E. coli BL21 (DE3) cells were transfected with the constructed vector and grown aerobically at 37°C in LB medium containing kanamycin at 40 μg/mL. Recombinant expression and purification of FN6K was carried out as previously described with one minor modification: the incubation for recombinant expression after isopropyl β‐D‐1‐thiogalactopyranoside addition was carried out at a lower temperature (18°C) for 24 h to reduce the amount of insoluble FN6K. A high yield of purified FN6K was achieved, 111 mg for a total of 610 U from 1 L of culture.…”

“…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.…”