Abstract
A Pseudomonas sp. soil strain, selected for its ability to grow on epsilon-(1-deoxyfructosyl) aminocaproic acid, was induced to express a membrane-bound enzymatic activity which oxidatively degrades Amadori products into free fructosamine. Apparent Km values for fructosyl aminocaproate, epsilon-fructosyl lysine, fructosyl glycine, and ribated lysine were 0.21 mM, 2.73 mM, 3.52 mM, and 1.57 mM, respectively. The enzyme was also active against alpha-fructosyl lysine and borohydride-reduced Amadori product, weakly active with ribated and glycated polylysine, and inactive with reducing sugars, amino acids, and glycated proteins. The enzymatic activity was highest at pH 6.5 and 25 degrees C in 0.1 M sodium phosphate, while over 80% of the activity was lost above 65 degrees C. Complete inhibition was observed by HgCl2, NaN3, and NaCN suggesting a role for SH groups and copper in the enzymatic activity. The reaction products were characterized by 1H NMR, 13C NMR, and GC/MS and found to correspond to 1-deoxy-1-aminofructose, i.e. free "fructosamine," and adipic acid. Confirmation of the free fructosamine structure was based on the complete spectroscopic identity of the borohydride reduction product with commercially available glucamine (1-amino-1-deoxyglucitol). The new enzyme is provisorily classified as fructosyl N-alkyl amino acid oxidase (EC 1.5.3) (fructosyl-amino acid:oxygen oxidoreductase) and may thus belong to a novel class of "Amadoriases" which deglycate Amadori products oxidatively. In contrast, however, the new enzyme acts on the alkylamine bond rather than the ketoamine bond of the Amadori product.
Highlights
A Pseudomonas sp. soil strain, selected for its ability to grow on E-(l-deoxyfructosyl) aminocaproic acid, was induced to express a membrane-bound enzymatic activity which oxidatively degrades Amadori products into free fructosamine
That protein had no Amadori product degrading activity. Searching for such activity, we describe, in this strain, the presence of membrane-bound enzymatic activity which decomposes Amadori products into free fructosamine and adipic acid under utilization of oxygen
Soil Strain-A preliminary experiment revealed that incubation of Pseudomonas sp. soil strain extract with lysine-Sepharose glycated with [14CJglucose was followed by release of radioactivity into the supernatant, suggesting the possible presence of enzymatic activity
Summary
BtmSA, N,O-bisCtrimethylsilyl)acetamide; fructosyl aminocaproate, e-Cl-deoxyfructosyl) aminocaproic acid; fructosyl glycine, I-deoxyfructosylglycine;fructosyllysine, o-z-Boc-e-Cldeoxyfructosyl) lysine; ribated lysine, a-t-Boc-e-Cl-deoxyribosyl) lysine; HPLC, high performance liquid chromatography; TTC, triphenyltetrazolium chloride. Identification and Purification ofEnzymatic Degradation ProductsFor identification of the product, the reaction mixture contained 1.5 ILmol of sodium phosphate buffer (pH 8.0), approximately 8 mg of washed ammonium sulfate-precipitated soil organism extract, and 13.7 umol of fructosyl aminocaproate in a total volume of 170 ILL The reaction was performed at 25°C or 37 "C for 5 days. For confirmation and quantitation of adipic acid as the enzymatic split product of fructosyl aminocaproate, the material obtained after 5 days of incubation was subjected to TLC, as described before, and GCIMS with Electron Impact ionization (70 eV). Standard adipic acid (1 mg) and desalted dry experimental samples from 180 ILl of enzyme supernatant were dissolved in 1.0 ml of methanol and evaporated three times to remove excess water. 240 180 with 50 mg of NaBH. in 1 ml of 0.01 N NaOH for 60 min, acidified with 150 ILl of 6 N HCI, treated three times with methanolic HCI to remove borate salts, and taken up in 2 ml of H 2 0 from which 50 ILl were injected into the HPLC system
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