Peptidic Tryptophan Halogenation by a Promiscuous Flavin-Dependent Enzyme

Area of Science:

• Biochemistry
• Enzymology
• Synthetic Biology

Background:

• Enzymatic modifications of amino acids are crucial but often limited by enzyme specificity.
• Flavin-dependent halogenases (FDHs) typically modify specific peptides or peptide termini.
• The narrow substrate scope and cofactor requirements of known FDHs limit their synthetic applications.

Purpose of the Study:

• To characterize ChlH, a novel flavin-dependent halogenase from the chlorolassin biosynthetic gene cluster.
• To investigate the substrate scope and catalytic activity of ChlH.
• To explore the potential of ChlH for broad peptide and protein modification.

Main Methods:

• Enzyme characterization of ChlH.
• Scanning mutagenesis of the substrate peptide ChlA.
• Molecular dynamics simulations.
• Halogenation assays on diverse peptide substrates, including RiPPs and pharmacologically relevant peptides.
• Cell-free biosynthetic assays.

Main Results:

• ChlH, unlike other FDHs, efficiently halogenates internal tryptophan (Trp) residues in peptides, in addition to N- and C-terminal Trp.
• Mutagenesis and simulations revealed insights into substrate recognition and specificity, explaining limitations in native chlorolassin biosynthesis.
• Wild-type ChlH demonstrated broad applicability, successfully halogenating various peptide and protein substrates.
• Cell-free assays elucidated ChlH's substrate preferences, highlighting its promiscuity.

Conclusions:

• ChlH exhibits a unique and broad substrate specificity among FDHs, enabling modification of internal Trp residues in diverse peptide sequences.
• The promiscuity of ChlH suggests significant potential for its application in modifying a wide range of peptide and protein substrates.
• This enzyme represents a valuable tool for synthetic biology and biotechnology, facilitating novel peptide modifications.