ISOLATION, PURIFICATION AND STRUCTURE OF EXOCHELIN MS, THE EXTRACELLULAR SIDEROPHORE FROM MYCOBACTERIUM-SMEGMATIS

The extracellular siderophore from Mycobacterium smegmatis, exochelin MS, was isolated from iron-deficiently grown cultures and purified to >98% by a combination of ion-exchange chromatography and h.p.l.c. The material is unextractable into organic solvents, is basic (pI = 9.3-9. 5), has a lambda(max) at 420 nm and a probable K-s for Fe3+ of between 10(25) and 10(30). Its structure has been determined by examination o f desferri- and ferri-exochelin and its gallium complex. The methods u sed were electrospray-m.s. and one- and two-dimensional (NOESY, DQF-CO SY and TOCSY) H-1 n.m.r. The constituent amino acids were examined by chiral g.l.c analysis of N-trifluoroacetyl isopropyl and N-pentafluoro propionyl methyl esters after hydrolysis, and reductive HI hydrolysis, of the siderophore. The exochelin is a formylated pentapeptide: N-(de lta-N-formyl,delta N-hydroxy-R-ornithyl)-beta-alaninyl-delta N-hydroxy -R-ornithinyl-R-allo-threoninyl-delta N-hydroxy-S-ornithine. The linka ges involving the three ornithine residues are via their delta N(OH) a nd alpha-CO groups leaving three free alpha-NH2 groups. Although there are two peptide bonds, these involve the three R (D)-amino acids. Thu s the molecule has no conventional peptide bond, and this suggests tha t it will be resistant to peptidase hydrolysis. The co-ordination cent re with Fe3+ is hexadenate in an octahedral structure involving the th ree hydroxamic acid groups. Molecular modelling shows it to have simil ar features to other ferric trihydroxamate siderophores whose three-di mensional structures have been established. The molecule is shown to h ave little flexibility around the iron chelation centre, although the terminal (Orn-3) residue, which is not involved in iron binding except at its delta N atom, has more motional freedom.