Stuffed Epimerase Domains of Pyochelin Biosynthesis are Defunct Methyltransferases

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Chemistry

Background:

• Bacteria and fungi synthesize nonribosomal peptides (NRPs) using nonribosomal peptide synthetases (NRPSs).
• NRPs often contain D-amino acids, conferring unique chemical properties and stability.
• 2-Hydroxyphenylthiazoline natural products were proposed to utilize noncanonical,
• stuffed
• epimerase domains within NRPS adenylation domains for stereochemical inversion.

Purpose of the Study:

• To investigate the proposed catalytic activity of
• stuffed
• epimerase domains in 2-hydroxyphenylthiazoline natural product biosynthesis.
• To determine the mechanism of D-amino acid incorporation in these specific NRPs.

Main Methods:

• Synthesized substrate and product analogs of 2-hydroxyphenylthiazoline siderophores.
• Examined adenylation-epimerase didomains from *Pseudomonas aeruginosa* (pyochelin) and *Streptomyces venezuelae* (watasemycin).
• Compared enzymatic activity with homologous enzymes lacking the
• stuffed
• epimerase domain, such as from *Pseudomonas protegens* (enantiopyochelin).

Main Results:

• Enzymes exhibited adenylation activity, but no enzymatic epimerase activity was detected.
• Spontaneous racemization occurred for 2-hydroxyphenylthiazoline ethyl ester analogs and isolated intermediates.
• The presence or absence of the
• stuffed
• epimerase domain did not correlate with the stereochemistry of the final product.

Conclusions:

• The noncanonical
• stuffed
• epimerase domains in 2-hydroxyphenylthiazoline natural products are catalytically defunct.
• These domains likely function as methyltransferases or represent evolutionary remnants.
• Stereochemical inversion in these pathways occurs through a non-enzymatic mechanism.