Purification and characterization of ascamycin-hydrolysing aminopeptidase from Xanthomonas citri.
A nucleoside antibiotic, ascamycin (9-beta-[5'-0-(N-L-alanyl) sulphamoyl-D-ribofuranosyl]-2-chloroadenine), has a selective antibacterial activity against Xanthomonas species. When ascamycin was dealanylated, dealanylascamycin showed a broad antibacterial activity against various Gram-negative and Gram-positive bacteria. Xanthomonas citri is susceptible to ascamycin by virtue of the ascamycin-dealanylating enzyme on the cell surface [Osada & Isono (1985) Antimicrob. Agents Chemother. 27, 230-233]. The enzyme (Xc aminopeptidase) was purified from X. citri cells by successive DEAE-cellulose, chromatofocusing and Sephadex G-100 column chromatography to a homogeneous state. The purified enzyme exhibited a single band with an Mr of 38 000 in SDS/polyacrylamide-gel electrophoresis. Gel filtration on a calibrated column indicated a similar Mr value. The isoelectric point of the enzyme was 5.7. The enzyme catalysed the hydrolysis of the alanyl group of ascamycin and liberated alanine from the sulphamoyl nucleoside. The enzyme also catalysed the hydrolysis of L-proline beta-naphthylamide and L-alanine beta-naphthylamide. The optimal pH and temperature for enzyme activity were pH 7.5-8.0 and 35-40 degrees C respectively. The enzyme was inhibited by thiol-enzyme inhibitors (i.e. rho-chloromercuribenzoate and N-ethylmaleimide), but was not affected by various naturally occurring aminopeptidase inhibitors (i.e. amastatin, bestatin, pepstatin and leupeptin). Mn2+ and Mg2+ activated the enzyme, whereas Cu2+, Zn2+ and Cd2+ were inhibitory.
D015762:2-Chloroadenosine / Q000031:analogs & derivatives; D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000626:Aminopeptidases; D002413:Cations, Divalent / Q000494:pharmacology; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008970:Molecular Weight; D013379:Substrate Specificity; D014974:Xanthomonas / Q000201:enzymology*
D002413:Cations, Divalent; D015762:2-Chloroadenosine; C035935:5'-sulfamoyl-2-chloroadenosine; C042369:ascamycin; D000626:Aminopeptidases; C048227:ascamycin aminopeptidase; D000241:Adenosine