Kozo Hirokawa

Senescence marker protein-30 is a unique enzyme that hydrolyzes diisopropyl phosphorofluoridate in the liver

Senescence marker protein‐30 (SMP30) was originally identified as a novel protein in the rat liver, the expression of which decreases androgen‐independently with aging. We have now characterized a unique property of SMP30, the hydrolysis of diisopropyl phosphorofluoridate (DFP), which is similar to the chemical warfare nerve agents sarine, soman and tabun. Hydrolysis of DFP was stimulated equally well by 1 mM MgCl2, MnCl2 or CoCl2, to a lesser extent by 1 mM CdCl2 but not at all by 1 mM CaCl2. No 45Ca2+‐binding activity was detected for purified SMP30, suggesting that SMP30 is not a calcium‐binding protein, as others previously stated. Despite the sequence similarity between SMP30 and a serum paraoxonase (PON), the inability of SMP30 to hydrolyze PON‐specific substrates such as paraoxon, dihydrocoumarin, γ‐nonalactone, and δ‐dodecanolactone indicate that SMP30 is distinct from the PON family. We previously established SMP30 knockout mice and have now tested DFPase activity in their livers. The livers from wild‐type mice contained readily detectable DFPase activity, whereas no such enzyme activity was found in livers from SMP30 knockout mice. Moreover, the hepatocytes of SMP30 knockout mice were far more susceptible to DFP‐induced cytotoxicity than those from the wild‐type. These results indicate that SMP30 is a unique DFP hydrolyzing enzyme in the liver and has an important detoxification effect on DFP. Consequently, a reduction of SMP30 expression might account for the age‐associated deterioration of cellular functions and enhanced susceptibility to harmful stimuli in aged tissue.

Improved practical usefulness of firefly luciferase by gene chimerization and random mutagenesis

To improve the practical usefulness of the firefly luciferase, we performed gene chimerization between Photinus pyralis luciferase and a thermostable variant of Luciola cruciata luciferase. One chimeric luciferase showed low K(m) value for substrate ATP and similar stability to thermostable L. cruciata luciferase. We then introduced random mutations in the corresponding gene and screened for increased catalytic efficiency. Amino acid replacement of Thr219, Val239 and Val290 affected the kinetic parameters. Therefore, we combined these three mutations. One mutant, ABcT219I,V239I, showed high catalytic efficiency comparable to P. pyralis luciferase and high stability similar to thermostable L. cruciata luciferase. The pH-dependence of the bioluminescence emission spectra was also examined. In contrast to wild-type firefly luciferases characterized to date, the mutant did not show the pH-dependent red spectrum shift.

Enhancement of thermostability of fungal deglycating enzymes by directed evolution

Fructosyl peptide oxidases are valuable for the determination of glycoproteins such as hemoglobin A1c. For practical use in clinical diagnosis, we applied directed evolution to improve the thermostability of these enzymes. After two rounds of random mutagenesis and high-throughput screening, six thermostabilizing amino acid substitutions were identified. Therefore, site-directed and cassette mutageneses were applied to combine these six stabilizing mutations. The simultaneous mutants showed that the stabilizing effect of the amino acid replacement was cumulative. The sextuple mutant enzyme, R94K/G184D/F265L/N272D/H302R/H388Y, had a half-life of thermal inactivation at 50°C that was 79.8-fold longer than that of the parental fructosyl peptide oxidase. The thermostable variants also showed increased tolerance to digestion by a protease. The sextuple mutant enzyme did not lose its activity on incubation with neutral protease, while the wild-type enzyme almost completely lost its activity. Furthermore, three amino acid substitutions were introduced into another fructosyl peptide oxidase with a different substrate specificity. The half-life of inactivation at 50°C was 3.61-fold longer than that of the parent enzyme. These engineered fructosyl peptide oxidases will be useful for industrial application to clinical diagnosis.

Recombinant Agrobacterium AgaE-like Protein with Fructosyl Amino Acid Oxidase Activity

Agrobacterium tumefaciens AgaE-like protein had a similar sequence to that of a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1. To characterize the AgaE-like protein, we produced the enzyme in Escherichia coli, and purified it to homogeneity. The molecular mass of recombinant AgaE-like protein was 42 kDa on SDS-PAGE and 85 kDa on gel filtration. The protein acted on N-fructosyl valine and N-fructosyl glycine as substrates, but not on glycated protein or Nε-fructosyl lysine. Apparent Km for N-fructosyl valine and N-fructosyl glycine were 1.64 and 0.31 mM, respectively. The AgaE-like protein had maximum activity at pH 7.8 and 35°C in 0.1 M potassium phosphate, but more than 80% of its activity was lost at 40°C or more. In contrast to eukaryotic fructosyl amino acid oxidases, the AgaE-like protein contained noncovalently bound FAD as a cofactor and was inactive against Nε-fructosyl Nα-Z(benzyloxycarbonyl)-lysine. These characteristics were similar to a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1, suggesting that these prokaryotic enzymes comprise a new family of fructosyl amino acid oxidases.

An enzymatic method for the determination of hemoglobinA1C

Fructosyl peptide oxidase is a flavoenzyme that catalyzes the oxidative deglycation of N-(1-deoxyfructosyl)-Val-His, a model compound of hemoglobin (Hb)A1C. To develop an enzymatic method for the measurement of HbA1C, we screened for a proper protease using N-(1-deoxyfructosyl)-hexapeptide as a substrate. Several proteases, including Neutral protease from Bacillus polymyxa, were found to release N-(1-deoxyfructosyl)-Val-His efficiently, however no protease was found to release N-(1-deoxyfructosyl)-Val. Neutral protease also digested HbA1C to release N-(1-deoxyfructosyl)-Val-His, and then the fructosyl peptide was detected using fructosyl peptide oxidase. The linear relationship was observed between the concentration of HbA1C and the absorbancy of fructosyl peptide oxidase reaction, hence this new method is a practical means for measuring HbA1C.

Synthesis and inhibitory activity of substrate-analog fructosyl peptide oxidase inhibitors

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.

Crystallization and preliminary crystallographic analysis of two eukaryotic fructosyl peptide oxidases

Fructosyl peptide oxidase (FPOX) catalyses the oxidation of α-glycated dipeptides such as N(α)-(1-deoxy-D-fructos-1-yl)-L-valyl-L-histidine (Fru-ValHis) and is used in the diagnosis of diabetes mellitus. Here, two thermostable mutants of FPOX, CFP-T7 and EFP-T5M, were crystallized by the sitting-drop vapour-diffusion method. The crystal of CFP-T7 belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 110.09, c = 220.48 Å, and that of EFP-T5M belonged to the monoclinic space group P2(1), with unit-cell parameters a = 43.00, b = 230.05, c = 47.27 Å, β = 116.99°. The crystals of CFP-T7 and EFP-T5M diffracted to 1.8 and 1.6 Å resolution, respectively.