NOVEL DEGRADATION PATHWAY OF GLYCATED AMINO-ACIDS INTO FREE FRUCTOSAMINE BY A PSEUDOMONAS SP SOIL STRAIN EXTRACT

A Pseudomonas sp, soil strain, selected for its ability to grow on eps ilon-(1-deoxyfructosyl) aminocaproic acid, was induced to express a me mbrane bound enzymatic activity which oxidatively degrades Amadori pro ducts into free fructosamine, Apparent K-m values for fructosyl aminoc aproate, epsilon-fructosyl lysine, fructosyl glycine, and ribated lysi ne were 0.21 mM, 2.73 mM, 3.52 mM, and 1.57 mM, respectively, The enzy me was also active against alpha-fructosyl lysine and borohydride-redu ced Amadori product, weakly active with ribated and glycated polylysin e, and inactive with reducing sugars, amino acids, and glycated protei ns, 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 6 5 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 H-1 NMR, C-13 NMR, and GC /MS and found to correspond to 1-deoxy-1-aminofructose, i,e, free ''fr uctosamine,'' and adipic acid, Confirmation of the free fructosamine s tructure was based on the complete spectroscopic identity of the boroh ydride reduction product with commercially available glucamine (1-amin o-1-deoxyglucitol). The new enzyme is provisorily classified as fructo syl 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.