A shared mechanistic pathway for pyridoxal phosphate-dependent arginine oxidases.

The mechanism by which molecular oxygen is activated by the organic cofactor pyridoxal phosphate (PLP) for oxidation reactions remains poorly understood. Recent work has identified arginine oxidases that catalyze desaturation or hydroxylation reactions. Here, we investigate a desaturase from the Pseudoalteromonas luteoviolacea indolmycin pathway. Our work, combining X-ray crystallographic, biochemical, spectroscopic, and computational studies, supports a shared mechanism with arginine hydroxylases, involving two rounds of single-electron transfer to oxygen and superoxide rebound at the 4' carbon of the PLP cofactor. The precise positioning of a water molecule in the active site is proposed to control the final reaction outcome. This proposed mechanism provides a unified framework to understand how oxygen can be activated by PLP-dependent enzymes for oxidation of arginine and elucidates a shared mechanistic pathway and intertwined evolutionary history for arginine desaturases and hydroxylases.

Recombinant expression, antimicrobial activity and mechanism of action of tritrpticin analogs containing fluoro-tryptophan residues.

The increase in antibiotic-resistant bacterial infections has prompted significant academic research into new therapeutic agents targeted against these pathogens. Antimicrobial peptides (AMPs) appear as promising candidates, due their potent antimicrobial activity and their ubiquitous presence in almost all organisms. Tritrpticin is a member of this family of peptides and has been shown to exert a strong antimicrobial activity against several bacterial strains. Tritrpticin's main structural characteristic is the presence of three consecutive Trp residues at the center of the peptide. These residues play an important role in the activity of tritrpticin against Escherichia coli. In this work, a recombinant version of tritrpticin was produced in E. coli using calmodulin as a fusion protein expression tag to overcome the toxicity of the peptide. When used in combination with glyphosate, an inhibitor of the endogenous synthesis of aromatic amino acids, this expression system allowed for the incorporation of fluorinated Trp analogs at very high levels (>90%). The antimicrobial activity of the 4-, 5- and 6-fluoro-Trp-containing tritrpticins against E. coli was as strong as the activity of the native peptide. Similarly, the tritrpticin analogs exhibited comparable abilities to perturb and permeabilize synthetic lipid bilayers as well as the outer and inner membrane of E. coli. Furthermore, the use of 19F NMR spectroscopy established that each individual fluoro-Trp residue interacts differently with SDS micelles, supporting the idea that each Trp in the original tritrpticin plays a different role in the perturbing/permeabilizing activity of the peptide. Moreover, our work demonstrates that the use of fluoro-Trp in solvent perturbation 19F NMR experiments provides detailed site-specific information on the insertion of the Trp residues in biological membrane mimetics. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.

Correction: An engineered biosynthetic-synthetic platform for production of halogenated indolmycin antibiotics.

[This corrects the article DOI: 10.1039/D0SC05843B.].

An engineered biosynthetic-synthetic platform for production of halogenated indolmycin antibiotics.

Indolmycin is an antibiotic from Streptomyces griseus ATCC 12648 with activity against Helicobacter pylori, Plasmodium falciparum, and methicillin-resistant Staphylococcus aureus. Here we describe the use of the indolmycin biosynthetic genes in E. coli to make indolmycenic acid, a chiral intermediate in indolmycin biosynthesis, which can then be converted to indolmycin through a three-step synthesis. To expand indolmycin structural diversity, we introduce a promiscuous tryptophanyl-tRNA synthetase gene (trpS) into our E. coli production system and feed halogenated indoles to generate the corresponding indolmycenic acids, ultimately allowing us to access indolmycin derivatives through synthesis. Bioactivity testing against methicillin-resistant Staphylococcus aureus showed modest antibiotic activity for 5-, 6-, and 7-fluoro-indolmycin.

Emergence of oxygen- and pyridoxal phosphate-dependent reactions.

Pyridoxal 5'-phosphate (PLP) is an organic cofactor employed by ~ 4% of enzymes. The structure of the PLP cofactor allows for the stabilization of carbanions through resonance. A small number of PLP-dependent enzymes employ molecular oxygen as a cosubstrate. Here, we review the biological roles and possible mechanisms of these enzymes, and we observe that these enzymes are found in multiple protein families, suggesting that reaction with oxygen might have emerged de novo in several protein families and thus could be directed to emerge again through laboratory evolution experiments.