Kanamycin-neomycin phosphate transferase

The reaction mechanism of kanamycin-neomycin phosphate transferase has been studied in detail, and the result was sound enough to permit prediction of the structures of derivatives active in inhibiting the growth of resistant organisms. The compounds thus synthesized are proving useful in the treatment of resistant infections, and are becoming more and more valuable as probes for further analysis of the biochemical mechanism of resistance. 

Escherichia coli K12 ML1629, . coU K12 ML1410 R81, and E. coli K12 J5 Rll-2 showed similar resistant patterns to aminoglycosidic antibiotics, and the S-100 fraction contained kanamycin-neomycin phosphate transferase I. This enzyme solution also inactivated lividomycins A and B (6 and 7) in the presence of adenosine 5 -triphosphate. A crude powder of the inactivated lividomydn A extracted by column chroma-... 

Partial purification of the lividomycin-inactivating enzyme was attempted by Mitsuhashi and coworkers, who briefly described their discovery that the enzyme obtained by fractionation with ammonium sulfate and column chromatography on Sephadex G-lOO inactivates lividomycins A and B, but not kanamycin A, indicating involvement of two different enzymes in the phosphorylation of lividomycin and kanamycin. However, it was definitely proved by H. Umezawa and coworkers that kanamycin-neomycin phosphate transferase I phosphorylates the 5"-hydroxyl group of lividomycins. The observation by Mitsuhashi and coworkers was probably occasioned by instability of the enzyme and the higher activity of the enzyme in phosphorylating lividomycins than in phosphorylating kanamycin A. 

Kanamycin-neomycin phosphate transferase I was successfully purified by affinity chromatography with lividomycin A-Sepharose 4B. Livi-... 

Independently, Umezawa and coworkers and Brzezinska and Davies" found another enzyme, designated kanamycin phosphate transferase II or kanamycin-neomydn phosphate transferase II, in E. coli canning R factor, and it was found by the former authors that this enz)one also catalyzes the transfer of a phosphate group from adenosine 5 -triphos-phate to the 3 -hydroxyl group of the kanamycins, paromamine, neamine, neomycin, ribostamydn, and the butirosins. Therefore, kanamycin-neomycin phosphate transferase II differs from I in the following points II phosphorylates butirosin, but not lividomydn, whereas I phospho-rylates the latter, but not the former. 

Kanamycin-neomycin phosphate transferase II also requires magnesium ion for reaction, and its optimal pH is 6.6-7.S. It is more stable to heat than kanamycin-neomycin phosphate transferase I. It phosphorylates all of the kanamycins, 4-0-(6-amino-6-deoxy-a-D-glucopyran-osyl)-6-0- -D-glucopyranosyl-2-deoxystreptamine, 4-0-(6-amino-6-deoxy-... 

D-glucopyranosyl)-2-deoxystreptamine, neamine, paromamine, neomycin, paromomycin, ribostamycin, butirosin A, and butirosin B. Thus, the 3-amino-3-deoxy-D-gIucose moiety in the kanamycins and the d-ribose moiety in the butirosins are not involved in the reaction. Similar structural requirements for inhibitors of kanamycin-neomycin phosphate transferase of Pseudomonas aeruginosa, as later described (see p. 201), were also observed for kanamycin-neomycin phosphate transferase II. Phosphorylation of butirosin A was competitively inhibited by one tenth the concentration of 3, 4 -dideoxykanamycin B. 

The involvement of kanamycin-neomycin phosphate transferase II in the mechanism of resistance was demonstrated by the fact that Escherichia coli KI2 JR66/W677 is inhibited by 3, 4 -dideoxybutirosin B, 3, 4 -dideoxyribostamycin,- 3, 4 -dideoxyneamine, and lividomycins that do not undergo this enzyme reaction. 

Kanamycin-Neomycin Phosphate Transferase in Pseudomonas aeruginosa... 

Kanamycin-neomycin phosphate transferase in this strain was partially purified by Umezawa and coworkers. 

The strain of Pseudomonas aeruginosa He is not resistant to livido-mycins, and kanamycin-neomycin phosphate transferase obtained from this strain is more stable than kanamycin-neomycin phosphate transferase I. Therefore, kanamycin-neomycin phosphate transferase of Ps. aeruginosa Hg is more closely related to kanamycin-neomycin phosphate transferase II in E. coli carrying R factor, as already described (see p. 196). A similar kanamycin-neomycin phosphate transferase was also observed in another strain, Ps. aerugjinosa TK-157. 

Cross-resistance between kanamycins and lividomycins is frequently observed in staphylococci, suggesting the presence of kanamycin-neomycin phosphate transferase I. However, phosphorylation of butiro-sins or lividomycins by a staphylococcus enzyme has not yet been studied. 

Lividomycins lack a 3 -hydroxyl group that could undergo the reaction of a kanamycin-neomycin phosphate transferase, but the 5"-hydroxyl group is phosphorylated by kanamycin-neomycin phosphate transferase... 

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