Broad substrate scope C-C oxidation in cyclodipeptides catalysed by a flavin-dependent filament

Area of Science:

• Biochemistry
• Enzymology
• Natural Product Biosynthesis

Background:

• Cyclic dipeptides possess anticancer and antimicrobial activities, often mediated by oxidative modifications.
• Cyclodipeptide oxidases (CDOs) catalyze key C-C bond oxidations but remain underexplored due to complex structures and unknown mechanisms.

Purpose of the Study:

• To elucidate the structure and mechanism of the cyclodipeptide oxidase from Nocardiopsis dassonvillei (NdasCDO).
• To characterize NdasCDO's role in nocazine natural product biosynthesis.
• To expand understanding of flavin-dependent oxidase enzymes.

Main Methods:

• X-ray crystallography to determine enzyme structure.
• Biochemical assays to assess substrate specificity and reaction conditions.
• Pre-steady-state kinetics and kinetic isotope effect studies to probe the catalytic mechanism.

Main Results:

• NdasCDO forms filaments in solution with a covalently bound flavin mononucleotide (FMN) cofactor.
• The enzyme processes various cyclic dipeptides distributively and is optimal at high pH.
• A radical intermediate is formed, and FMN regeneration is the rate-limiting step.

Conclusions:

• NdasCDO exhibits unique filament formation and a complex catalytic mechanism involving radical intermediates.
• The enzyme's promiscuity and optimal conditions position it as a potential biocatalyst.
• This work expands the known diversity of FMN-dependent oxidases to include filamentous enzymes.