Evidence that a deferrioxamine B degrading enzyme is a serine protease

Abstract

Siderophores are organic biomolecules synthesized by a wide variety of microbes. The molecules sequester ferric ion from environments where it is present at extremely low concentrations. Siderophores are of consequence with respect to microbial nutrition, pathogenicity, virulence, and microbe-plant interactions. How siderophores are degraded and returned to the carbon and nitrogen cycles is not well understood. The catalytic activity of an enzyme from a bacterium that degrades the siderophore deferrioxamine B has been examined. While the degradation of deferrioxamine B is sensitive to sulfhydryl and metal moiety inhibitors, the data presented is most consistent with the hypothesis that the enzyme uses a hydroxyl moiety (serine peptidase) to catalyze the degradation of deferrioxamine B. If sulfhydryl and metal inhibitors are simultaneously present at concentrations that when alone only partially inhibit the enzyme, the enzyme is unable to catalyze deferrioxamine B dissimilation. Analysis of the inhibitor experiments conducted led to the conclusion that the deferrioxamine B degrading enzyme is a serine-peptidase-like enzyme that needs calcium ions and sulfhydryl groups to be fully activated or stabilized. The knowledge of the catalytic moieties of the enzyme will be exploited to purify the enzyme.

Degradation Pathway and Generation of Monohydroxamic Acids from the Trihydroxamate Siderophore Deferrioxamine B

“…M. loti was aerobically grown in a mineral salts medium supplemented with 5 ml of vitamin solution liter Ϫ1 and 0.1 to 0.3% of DFB as described previously (4,17,39). DFB was supplied as mesylate salt by Novartis Pharmaceuticals Corporation (Summit, N.J.).…”

“…DFB was supplied as mesylate salt by Novartis Pharmaceuticals Corporation (Summit, N.J.). DFB hydrolase was harvested from M. loti as previously described (17,39). Briefly, late-logarithmic-phase cells were harvested, and cell-free enzyme extract was obtained via sonication to approximately 90% breakage.…”

“…Much research has been conducted to investigate the biosynthesis, iron chelation, iron assimilation, and genetics which allow microbes to acquire iron via siderophores. Far less attention, however, has been given to the study of how siderophores are mineralized and returned to the carbon and nitrogen cycles.Three siderophore-degrading microbes, a pseudomonad (33-35), Azospirillum irakense (36, 37), and Mesorhizobium loti (4,7,17,39), catabolize siderophores concomitant with their growth. Neilands and colleagues (33-35) observed that their microbe, named Pseudomonas FC1, degraded the hydroxamate siderophores ferrichrome, ferrichrome A, and coprogen, with ferrichrome A being the most facile to degrade.…”

“…Three siderophore-degrading microbes, a pseudomonad (33)(34)(35), Azospirillum irakense (36,37), and Mesorhizobium loti (4,7,17,39), catabolize siderophores concomitant with their growth. Neilands and colleagues (33)(34)(35) observed that their microbe, named Pseudomonas FC1, degraded the hydroxamate siderophores ferrichrome, ferrichrome A, and coprogen, with ferrichrome A being the most facile to degrade.…”

“…Unlike for the A. irakense isolate, however, the ferric analogue of DFB, ferrioxamine B (FB), serves as neither an iron source nor a carbon source for M. loti (7). M. loti also differs from A. irakense in that no DFB degradation products are released to the culture medium and such products were noted only when a cell extract, containing the cytosolic, DFB-degrading enzyme(s) (trivially named DFB hydrolase [4,5,17,39]) was given DFB as a substrate.…”