fructosylvaline and fructosyl-lysine

Fructosyl peptide oxidases (FPOXs) play a crucial role in the diagnosis of diabetes. Their main function is to cleave fructosyl amino acids or fructosyl peptides into glucosone and the corresponding amino acids/dipeptides. In this study, the substrate-analog FPOX inhibitors 1a-c were successfully designed and synthesized. These inhibitors mimic N(α)-fructosyl-L-valine (Fru-Val), [N(α)-fructosyl-L-valyl]-L-histidine (Fru-ValHis), and N(ε)-fructosyl-L-lysine (εFru-Lys), respectively. The secondary nitrogen atom in the natural substrates, linking fructose and amino acid or dipeptide moieties, was substituted in 1a-c with a sulfur atom to avoid enzymatic cleavage. Kinetic studies revealed that 1a-c act as competitive inhibitors against an FPOX obtained from Coniochaeta sp., and Ki values of 11.1, 66.8, and 782 μM were obtained for 1a-c, respectively.

Topics: Amino Acid Oxidoreductases; Ascomycota; Dose-Response Relationship, Drug; Enzyme Inhibitors; Kinetics; Lysine; Molecular Structure; Structure-Activity Relationship; Valine

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.

Topics: Amino Acid Substitution; Catalytic Domain; Escherichia coli; Fructosamine; Lysine; Metabolic Engineering; Models, Molecular; Mutagenesis, Site-Directed; Mutant Proteins; Phosphotransferases; Protein Engineering; Substrate Specificity; Valine

Docking models of fructosyl amine oxidase (FAOD) from the marine yeast Pichia N1-1 (N1-1 FAOD) with the substrates fructosyl valine (f-Val) and fructosyl-(epsilon)N-lysine (f-(epsilon)Lys) were produced using three-dimensional protein model as reported previously (Miura et al., 2006, Biotechnol. Lett., 28, 1895-1900). The residues involved in recognition of substrates were proposed, particularly Asn354, which interacts closely with f-(epsilon)Lys, but not with f-Val. Substitution of Asn354 to histidine and lysine simultaneously resulted in an increase in activity of f-val and a decrease in activity of f-(epsilon)Lys and thus, increasing the specificity for f-Val from 13- to 19-fold. In addition to creating two mutant FAODs with great potential for the measurement of glycated hemoglobin, we have provided the first structural model of substrate binding with eukaryotic FAOD, which is expected to contribute to further investigation of FAOD.

Topics: Amino Acid Oxidoreductases; Asparagine; Bacillus; Buffers; Glycated Hemoglobin; Lysine; Models, Biological; Mutagenesis, Site-Directed; Mutation; Pichia; Recombinant Proteins; Sarcosine Oxidase; Substrate Specificity; Valine

Fructosyl-amino acid oxidase (FOD-F) from Fusarium oxysporum f. sp. raphani (NBRC 9972) is the enzyme catalyzing the oxidative deglycation of fructosyl-amino acids such as N(epsilon)-fructosyl N(alpha)-benzyloxycarbonyl-lysine (FZK) and fructosyl valine (FV), which are model compounds of the glycated proteins in blood. Wild-type FOD-F has high activities toward both substrates. We obtained a mutant FOD-F, which reacts with FZK but not with FV by random mutagenesis. One amino-acid substitution (K373R) occurred in the mutant FOD-F. In addition to K373R, K373W, K373M, K373T, and K373V, which were selected for optimization of the substitution at position K373, were purified and characterized. Kinetic analysis showed that the catalytic turnover for FV greatly decreased, whereas that for FZK did not. In consequence, the specificities toward FZK were increased in the mutant FOD-Fs. The relation between the substrate specificity of the mutant FOD-Fs and the position of the carboxyl group of the substrates was demonstrated using a series of the substrates having the carboxyl group at the different position. The mutant FOD-Fs are attractive candidates for developing an enzymatic measurement method for glycated proteins such as glycated albumin in serum. This study will be helpful to establish an easier and rapid clinical assay system of glycated albumin.

Topics: Amino Acid Oxidoreductases; Amino Acid Sequence; Amino Acid Substitution; Fusarium; Kinetics; Lysine; Molecular Sequence Data; Mutagenesis; Mutation, Missense; Substrate Specificity; Valine

Our fungal culture collection was screened for fructosyl peptide oxidase, an enzyme that could be used for the determination of glycated hemoglobin in diabetic subjects with hyperglycemia. Fructosyl peptide oxidases were found in strains of eight genera: Achaetomiella, Achaetomium, Chaetomium, Coniochaeta, Eupenicillium, Gelasinospora, Microascus and Thielavia. By their substrate specificity toward N(alpha)-fructosyl valyl-histidine (alpha-keto-amine) and N(epsilon)-fructosyl lysine (epsilon-keto-amine), fructosyl peptide oxidases could be categorized into two groups: (1) enzymes that oxidize both alpha-keto-amine and epsilon-keto-amine, and (2) enzymes that preferably oxidize alpha-keto-amine. A fructosyl peptide oxidase from Achaetomiella virescens ATCC 32393, active toward both N(alpha)-fructosyl valyl-histidine and N(epsilon)-fructosyl lysine, was purified to homogeneity and characterized. The enzyme was monomeric ( M(r)=50,000), was most active at 40 degrees C and pH 8.0, and had a covalently bound flavin as a prosthetic group. Apparent K(m) values for N(alpha)-fructosyl valyl-histidine and N(epsilon)-fructosyl lysine were 2.30 and 1.69 mM, respectively. N(alpha)-fructosyl valyl-histidine was consumed and the same molar amount of valyl-histidine was produced by the fructosyl peptide oxidase reaction. This enzyme could be useful for the measurement of hemoglobin A(1C), the N-terminal valine residue of the beta-subunit of which is glycated.

Topics: Amino Acid Oxidoreductases; Chromatography, High Pressure Liquid; Glycated Hemoglobin; Histidine; Hydrogen-Ion Concentration; Lysine; Sordariales; Substrate Specificity; Temperature; Valine

An enzyme-sensor system with flow-injection analysis (FIA) has been developed for the detection of fructosyl amine compounds; the sensor utilizes fructosyl amine oxidase isolated from the marine yeast Pichia sp. N1-1 strain. With this FIA system 0.2 to 10 mmol L(-1) fructosyl valine can be determined. The sensor is approximately five times more sensitive to fructosyl valine, a model compound for glycated hemoglobin HbA1c, than to N(epsilon)-fructosyl lysine, a model compound for glycated albumin. This FIA system can also be used to detect fructosyl dipeptides. The operational stability of the sensor enabled more than 120 consecutive sample injections over a period of approximately 20 h.

Topics: Amino Acid Oxidoreductases; Biosensing Techniques; Calibration; Enzymes, Immobilized; Glycated Hemoglobin; Glycoproteins; Humans; Lysine; Valine