Direct α-Hydroxy Acid Loading onto a Bacterial Thiotemplate Assembly Line via a Multienzyme Gateway

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Nonribosomal peptide synthetases (NRPSs) incorporate diverse building blocks, including alpha-hydroxy acids, into peptides.
• Malleicyprol, a virulence factor from Burkholderia pseudomallei (BP) pathogens, contains the unique trigonic acid warhead.
• The mechanism for NRPS loading of trigonic acid was previously unknown due to an incomplete NRPS module.

Purpose of the Study:

• To elucidate the mechanism of trigonic acid loading onto NRPS.
• To identify the enzymes responsible for activating and translocating trigonic acid.
• To expand the synthetic biology toolbox for incorporating non-canonical building blocks.

Main Methods:

• Bioinformatics analysis
• Mutational studies of key enzymes
• Targeted metabolomics
• In vitro biochemical assays

Main Results:

• Two trans-acting enzymes, BurJ and BurH, are essential for trigonic acid loading.
• BurJ (adenylation-thiolation didomain enzyme) activates trigonic acid.
• BurH (FkbH-like protein with mutated phosphatase domain) translocates the activated trigonic acid thioester.
• This represents the first reported direct loading of an alpha-hydroxy acid onto a bacterial NRPS module.

Conclusions:

• A novel two-enzyme gateway facilitates the incorporation of trigonic acid into NRPS assembly lines.
• This finding provides insights into virulence factor biosynthesis in Burkholderia pathogens.
• The discovered mechanism expands possibilities for site-specific incorporation of non-canonical building blocks in synthetic biology.
• Understanding this pathway may lead to the development of anti-virulence therapeutics against BP infections.