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

So, you want to know about siderophore synthesis

Presented by:

Steven Backues

Brooks Maki

and

Donnie Berkholz “The Invisible Man”

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

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

Danoxamine

Composed of a linear series of three hydroxamic acids.

Composed of two major groups linked by succinic acid.

Biosynthesis of Siderophores

How it’s really done.

Hydroxamate Siderophores

Step 1: Ornithine N5-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 O2 and NADPH to give an N-O bond at the end of its side chain.

Step 2: N5-Transacylase

The nitrogen modified in this way is additionally attached to an acyl group carried by coenzyme A

This completes the hydroxamate prosthetic group

The Hydroxamate prosthetic group:

N

NH3+

H

COO-

HO

O

H3C

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.

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

Thiolation

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

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.

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.

Vibriobactin

OH

OH

O

N

NH

Me

O

N

HO

HO

O

N

MeO

NH

O OH

OH

The Cathechol Prosthetic Group

The cathechol prosthetic group is 2,3-dihydroxybenzoic acid, which is formed from chorismic acid

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

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

Norspermidine+cathecol

OH

OH

O

N

NH

Me

O

HN NH2

Yersiniabactin

OH

S

N

S

NH

MeMe S

N

Me

COOH

OH

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

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

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

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

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.

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