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

被引:72
作者
SHARMAN, GJ
WILLIAMS, DH
EWING, DF
RATLEDGE, C
机构
[1] UNIV HULL,DEPT APPL BIOL,KINGSTON HULL HU6 7RX,N HUMBERSIDE,ENGLAND
[2] UNIV CAMBRIDGE,CAMBRIDGE CTR MOLEC RECOGNIT,DEPT CHEM,CAMBRIDGE CB2 1EW,ENGLAND
[3] UNIV HULL,DEPT CHEM,KINGSTON HULL HU6 7RX,N HUMBERSIDE,ENGLAND
关键词
D O I
10.1042/bj3050187
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 of desferri- and ferri-exochelin and its gallium complex. The methods used were electrospray-m.s. and one- and two-dimensional (NOESY, DQF-COSY 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-pentafluoropropionyl methyl esters after hydrolysis, and reductive HI hydrolysis, of the siderophore. The exochelin is a formylated pentapeptide: N-(delta-N-formyl,delta N-hydroxy-R-ornithyl)-beta-alaninyl-delta N-hydroxy-R-ornithinyl-R-allo-threoninyl-delta N-hydroxy-S-ornithine. The linkages involving the three ornithine residues are via their delta N(OH) and alpha-CO groups leaving three free alpha-NH2 groups. Although there are two peptide bonds, these involve the three R (D)-amino acids. Thus the molecule has no conventional peptide bond, and this suggests that it will be resistant to peptidase hydrolysis. The co-ordination centre with Fe3+ is hexadenate in an octahedral structure involving the three hydroxamic acid groups. Molecular modelling shows it to have similar features to other ferric trihydroxamate siderophores whose three-dimensional structures have been established. The molecule is shown to have 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.
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页码:187 / 196
页数:10
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