Characterization of a Dual Function Peptide Cyclase in Graspetide Biosynthesis

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Graspetides are ribosomally synthesized peptides with complex macrocyclic structures formed by ATP-grasp enzymes.
• Group 11 graspetides, like prunipeptin, possess both macrolactone and macrolactam cross-links, requiring dual macrocyclization.
• Mechanistic details of ATP-grasp enzymes involved in dual macrocyclization remain largely uncharacterized.

Purpose of the Study:

• To reconstruct and investigate the biosynthesis of prunipeptin from *Streptomyces coelicolor*.
• To elucidate the enzymatic mechanisms of the PruA and PruB ATP-grasp macrocyclases.
• To gain insights into the structure-function relationships of these enzymes for potential engineering.

Main Methods:

• Reconstruction of prunipeptin biosynthesis using recombinant PruA and PruB macrocyclases.
• Kinetic analysis of PruB enzyme activity, including determination of kinetic parameters like *k*_cat.
• X-ray crystallography to determine the structure of PruB and site-directed mutagenesis to identify key residues.
• Computational modeling of the PruA/PruB cocomplex to understand substrate interactions.

Main Results:

• PruB displayed enzyme kinetics comparable to other graspetide cyclases, with enhanced catalytic efficiency potentially due to an ATP-regeneration system.
• The X-ray crystal structure of PruB showed unique features distinguishing it from Group 1 and 2 ATP-grasp enzymes.
• Site-directed mutagenesis confirmed the importance of specific residues, including the conserved DxR motif, for PruB's catalytic function.
• Computational modeling suggested a sequential mechanism where PruB likely initiates macrolactone formation on PruA.

Conclusions:

• This study provides significant mechanistic insights into the ATP-grasp enzymes PruA and PruB involved in Group 11 graspetide biosynthesis.
• The findings enhance the understanding of dual macrocyclization processes mediated by ATP-grasp enzymes.
• The detailed structural and mechanistic information offers a foundation for engineering these enzymes for novel biotechnological applications.