1. So, you want to know about siderophore synthesis Presented by: Steven Backues Brooks Maki and Donnie Berkholz “The Invisible Man”

2. Hydroxamic Acid Groups  N-Alkylation of O-substituted Hydroxamic acid.  Formation of an oxime from an aldehyde and a hydroxylamine. Followed by reduction and acylation  These derivatives allowed synthesis of several siderophores and their analogues

3. Alternative Methods  Oxidation of Lysine and OrnithineWith DMD (dimethyldioxarine)  Conversion of primary amines to imines, with oxidation of imines to oxaziridines. Hydrolysis leads to hydroxylamines.  D-Ferrichrome synthesized by this method

4. Danoxamine  Composed of a linear series of three hydroxamic acids.  Composed of two major groups linked by succinic acid.

7. Biosynthesis of Siderophores How it’s really done.

8. Hydroxamate Siderophores

9. Step 1: Ornithine N 5 - Oxygenase  The formation of the N-O bond is the first committed step in hydroxamate synthesis  Ornithine, an amino acid used in the urea cycle, is reacted with O 2 and NADPH to give an N-O bond at the end of its side chain.

10. Step 2: N 5- Transacylase  The nitrogen modified in this way is additionally attached to an acyl group carried by coenzyme A  This completes the hydroxamate prosthetic group

11. The Hydroxamate prosthetic group :

12. Step 3: Non-ribosomal Peptide Synthetase  This synthetase is a large complex with many subdomains, including an adenylation domain, a thiolation domain, and a condensation domain.

13. Adenylation  First, the hydroxamate is is activated by addition of an adenylate group at its C terminus  The source of the adenylate group is ATP, and the reaction occurs with production of pyrophosphate

14. Thiolation  The hydroxamate group is then transferred to the enzyme through the formation of a thioester linkage with displacement of the adenylate group.

15. Condensation  Finally, the hydroxamate group is attached to another molecule (perhaps another hydroxamate group, or else a growing chain) by the nucleophilic attack of an OH or NH from the chain on the S-linked carbonyl, displacing the sulfur.

16. Cathechols: Vibriobactin  Vibriobactin is a siderophore used by Vibrio cholerae  Its synthesis also involves a large, non- ribosomal peptide synthetase, and follows many of the same pathways as the synthesis of hydroxamate siderophores outlined above.

17. Vibriobactin

18. The Cathechol Prosthetic Group  The cathechol prosthetic group is 2,3- dihydroxybenzoic acid, which is formed from chorismic acid

19. Nonribosomal Peptide Synthetase  2,3-dihydrobenzoic acid then acts as a substrate for Vibirobactin Syntetase  It is first activated by adenylation, then transferred to the enzyme with formation of a thioester

20. Transfer to Norspermidine  This thioester complex then undergoes nucleophilic attack by a primary amine on norspermidine.  The norspermidine/cathechol complex goes on to react with two more cathechol prosthetic groups (these, however, attached by threonine derived linkages) to form the final siderophore

21. Norspermidine+cathecol

22. Yersiniabactin

23. Synthesis  Although it has neither hydroxamate nor cathechol groups, Yersiniabactin follows some of the same synthesis pathways, using a nonribosomal peptide sythetase that has clear homologies with, for example, vibriobactin sythetase

24. Beginnings  Synthesis begins with salicylic acid (2- hydroxy-benzoic acid)  This is activated by the attachment of an adenylate group, then loaded onto the enzyme by the formation of a thioester, as before

25. Elongation  At the same time, two cystines are also activated then loaded onto the same enzyme, also via a thioester linkage  Then, in the condensation/cyclization domain, the salicyate group is transferred onto one of the cystines, which is then cyclized.  This cyclization is an unusual property of this particular synthetaes

26. Completion  A second cystine is added, and also cyclized, and the resulting molecule undergoes the addition of a malonyl group, S- adenosylmethionine, and an additional cystine to complete the synthesis

27. Overview:  The use of a large, multidomain nonribosomal peptide synthetase was a common element of all of these syntheses.  All of these processes included the activation of a substrate by adenylation and the transfer to a thioester linkage with the enzyme, followed by condensation to form a longer chain. This is similar to the process followed in biosynthesis of fatty acids.

28. References  Roosenberg, J.M. and Miller, M.J. Total Synthesis of the Siderophore Danoxamine. J. Org. Chem. 2000 Vol. 65 No. 16. 4833 – 4838.  Lin, Y. and Miller, M.J. Synthesis of Siderophore Components by and Indirect Oxidation Method. J. Org. Chem. 1999 Vol. 64 No. 20. 7451 – 7458.  Gaspar, M., Grazina, R., Bodor, A., Farkas, E., and Santos, M.A. Macrocyclic tetraamine tris(hydroxamate) ligand. J. Chem Soc. 1999 799 – 806.  Duhme, A.K. Synthesis of two dioxomolybdenum complexes of a siderophore analogue. J. Chem. Soc. 1997 773 – 778.  Atkinson, A. Bacterial Iron Transport. Biochemistry. 1998. 15965 - 15973