The structure of a desferriferribaciin a postulated precursor of pyowrdins produced in iron-deficient cultures of i'seudomonasj7uorescens ATCC 13525 was elucidated using FAB-MS and 20 NMR techniques. II is a partly cyclic pe4xide containing a thirteen-membered ring, as well as an amkiine trpp amino-acid built up on tyrosine and 2,4 dir minobutyric acid. Whet! Pseudomonasfluorescens ATCC 13525 grows in iron deficient conditions, it excretes a large number of pyo\ erdins l1 possessing the same type of fluorescent chromophore derived from 2,3-diamino-6,7 dihydro>:yquinoline, together with a desferriferribactin which does not have a chromophore but which also chelates iron(II1). Maurer er a1.2 proposed a peptide structure of ten amino acids [Ser(2), Lys(3), N6 - OHOm(?), Tyr( I), Gly( l), Glu( I)] and two acetyl groups for the desferrifenibactin molecule. After reaction of the pepttde with dinitrofluorobenzene and hydrolysis, they characterized only DNP-glutamic acid and the NHE DNP-delivative from only one lysine molecule, and concluded that desfeniferribactin was cyclic.Philson and Llifias 3 corrected some of these data showing that desferrifetribactin has only two lysines, and that the two acetyl groups were in fact formyl groups. More recently Budziekiewicz and his group have reported some NhlR data on four desferriferribactins from different strains of fluorescent Pseuaimonas 45 as well as the strucure of a desferrifenibactin excreted by a strain of Pseudomonas aptata 5. They found that all contained 2,4-diaminobutyric acid (Dab) combined to tyrosine forming a cyclic amidine as in the pyoverdinsexcreted tgv two strains of fluorescent Pseudonwnus, namely pyoverdins ATCC 174006 and pyoverdins CCM 2798 (and!799)7 which respectively possess Gl@ab (or GlnCIHPMD) and &/Dab (or SerCIHPMD). They concluded by dansylation followed by hydrolysis and by NMR that glutarnic acid was at the N-terminus in the desfetrifen!bactins and that it is bound to ty-rosine by its y carboxyl group. However they did not report any structure n fr any sequence for desferrifenibactin ATCC 13525 and postulated that desferriferribactins were the biosynthetic precursors of pyoverdins45.Using 'he purification procedure we have previously described for pyoverdins, azotobactins and azoverdin 1 6-1 1 we were able to separate and isolate in pure state more than fifteen pyoverdins from the cultures of Pseudomonas jluorescens ATCC 13525 together with one desfeniferribactinl. Using FAB-MS as well as 2D NMR techniques, we establish here the structure of this latter as structure 2.Ferribactin has a MH+ at m/z 1231 (FAB-MS). Its corresponding free ligand, desferriferrlbactin, studied here by FitB-MS has the corresponding MI-I+ at m/z 1178 (All masses were assigned by counting and are therefore n :>minal).Acid h+olysis (6 M HCl, 1 mitt, 8o'C) resulted in two main fragments at m/z 1122 (hydrolysis of two formyl groups) and m/z 993 (loss of glutamic acid), this latter being consistent with a terminal position of this acid.Peracyl,uion with trifluoroacetic anhydride and acetic acid yielded a mixture of monotrifluoroacetyl derivatives having up to five acetyl groups (m/z 1484, 1442 and 1400) as well as hexaacetyl and pentaacetyl derivatives (m/z 1430,1388). No significant loss of formyl groups or formyl/acetyl exchange could be detected. Fragments were also found, which could be interpreted as a indicating that the trifluoroacetyl group (TFA) most probably acylated the glutamic acid amino group, and b (Table 1).Mild trethanolysis with a 1:l mixture of deuterated and undeuterated methanol, and HCI, followed by peracetylalion showed that deformylation took place, and that one free car-boxy1 group was then esterified (pentaacetqjl, m/z 1349-1346); tetraacetyl, m/z 1307-1304). Fragments c to f (Table 1) were also found, none of them contauting the esterified carboxyl group, and they are consistent with the sequence Ser.Lys-Gly. Reactioit with phenylisothiocyanate yielded a mono- (m/z 1313) and a di- (m/z 1448) phenylthiourea derivatives, indicating the presence of two amino groups of differing reactivity, in agreement with the previous results of itiaurer et d2, and leading to the conclusion that 2,4diaminobutyrlc acid does not possess a free amine gr011p in the peptide.Table 1: As:ignment of ions in FAB-MS of peracyl desfemferribactin and the peracetylati products of very mild methanolysis| Code | m/z | Assignment | | | | ||------|---------|-------------------------------------------------------------------------|--|--|--|--|| a | 617,659 | [Glu-Tyr-DabSerNH2 - H20 + H] + 1 TFA + 1 and 2 Acetyls | | | | || b | 801,843 | ILys-Gly-Nformyl,NOHOm-Lys-Nfomyl.NOHOm-Ser -H20 + Hj + 2 and 3 Acetyls | | | | || | 874,916 | [Ser-Lys-Gly-OHOm-Lys-OHOm-SW -H20 + H] + 3 and 4 Acetyls | | | | || : | 787,829 | ILys-Gly-OHOm-Lys-OHOm-Ser -H20 + H] + 3 and 4 Acetyls | | | | || e | 659,701 | [Gly-OHOm-Lys-OHOm-Ser -H20 + H] + 3 and 4 Acetyls | | | | || f | 602,661 | [OHOm-Lys-OHOm-Ser -H20 + H] + 3 and 4 Acetyk | | | | |Upon acidification under the Edman conditions, the loss of glutamic acid hydantoin from the di-derivative (m/z 1448) is, aI most, very minor (very weak peak at m/z 1183): together with the loss of water to mass 1430, this is consistent with the proposed desfexriferribactin sequence.From these data it could be concluded that desferriferribactin has the following sequence : Glu-(Tyr/Dab)-Ser-Lys-Gly-(OHOrn,Lys,OHOrn,Ser).2D NMR spectra showed that all the amino acids common to pyoverdin Pf ATCC 13525 and desferriferribactin have similar chemical shifts1. The HOHAHA assignment of all the amino acids signals showed a marked difference in the chemical shifts of the HE protons of the lysines (Table 2), which suggest, by comparison with previously reported data that Lys-5 is linked via its α amino group to the peptide chain, and Lys-8, via both its α and ε amino groups 1,7,10.Table 2: Assignment of the protons of desferriferribactin Pf ATCC 13525 at 280°K in H2O/([^2H]_3C)_3CO(^2H]$ , acidified to pH 3.0 by addition of CF3( OOH and using sodium [^2H_2]_6-trimethylsilylpropane sulfonate as an in| | NΗα | $CH\alpha$ | $CHB$ | CHY | $CH\delta$ | CHt | NΗω ||-------------------|------|------------|-------|-----------------------------|---------------|-----------|-----------|| $Glu-1$ | 7.95 | 3.68 | 2.09 | 2.46 | | | || $Tyr-2$ | 8.68 | 4.67 | 3.11 | 7.17 (H3, H5) 6.86 (H2, H6) | | | || Dab-3 | | 4.41 | 2.09 | 3.33 | | | 9.41-9.56 || Ser-4 | 8.96 | 4.34 | 3.87 | | | | || $Lys-5$ | 8.83 | 4.38 | 1.89 | 1.43 | 1.66-1.73 | 2.97 | 7.60 || | 8.54 | 3.94 | | | | | || Gly-6NºOHOm-7 | 8.17 | 4.39 | 1.74 | 1.58 | 3.55 | | || | 8.34 | 4.11 | 1.90 | $0.98 - 1.34$ | $1.47 - 1.56$ | 3.16-3.28 | 7.38 || Lys-8NoOHOm-9 | 8.41 | 4.21 | 1.75 | 1.64 | $3.55$ | | || $Ser-10$ | 8.49 | 4.44 | 3.87 | | | | |Formyl7.95 (cis) and 8.25 (trans) 0.5:1.52D ROESY NMR techniques completed these findings; after having assigned all the protons of the amino acids constituting the peptide, the spectra were determined in two different conditions and gave a number of cross peaks which yielded the final sequence of desferriferribactin Pf ATCC 13525. At 300°K using a ${}^{2}H_{2}O/H_{2}O$ 1:9 mixture and a 300 ms mixing time, the connectivities between the protons CHi, NHi and $NH_{i+1}$ of the amino acids represented by arrows in solid lines in 3a, gave the following partial sequences: Glu(1)-Tyr(2)/Dab(3)-Ser(4) and Lys(5)-Gly(6)-OHOrn(9)-Lys(8)-OHOrn(9)-Ser(10). In addition the two formyl groups which resonate as a set of two signals of unequal intensity at 7.90-7.91 and 8.26-8.28 ppm (cis and trans with a ratio of 1.5:0.5) are each connected to the CHS protons of the corresponding $N^0$. hydroxyomithine.At 280'K, '1 a mixture of H20/([2H]3C)3C-O[2H] 9:l acidified with CF3CO2H to pH 3.0 and a mixing time of 400 ms most of the connectivities were the same. The new cross peaks occurring in these conditions (represente,! by solid arrows in 3b) show in addition that in desferrifen-ibactin, Ser(l0) is bound to Lys(8) through its NHE in a similar fashion as in pyoverdins Pf ATCC 13525 1 and confirm the presence of the same 13 member-d ring. The complete sequence of desfenifenibactin ATCC 13525 isGlu(l)-T~I (2)/Dab(3)-Ser(4)-Lys(S)-Gly(6)-OHOm(7)-Lys~) ->er(lO). OHOrn(9) GC-MS *3f the O-methyl, N-heptafluorobutyryl esters on a L-Chirasil-Val column of a total hydrolyzate of desferriferr~bactin Pf ATCC 13525 confirmed the results already reported 2-4 and showed that all the amino acids common to this compound and pyoverdins ATCC 13525 1 have the same stereochemistry.The strLr:ture of desferriferribactin Pf ATCC 13525 is reported in 2. It is very similar to the structure of pyoverdins Pf ATCC 13525 l1 and is compatible with the plausible role of desferriferribactins as biogenetic precursors in the biosynthesis of the siderophores, tyrosine being oxidized to dopa which together with the tetrahydrop*timidine ring built up between tyrosine and 2.4~diaminobutyric acid yields the fluorescent chromophcr,e of pyoverdins