Effects of copper occupancy on the conformational landscape of peptidylglycine \u03b1-hydroxylating monooxygenase

Sweta Maheshwari,Richard E Mains,Katarzyna Rudzka,L Mario Amzel,Chelsey D Kline,Sandra B Gabelli,Betty A Eipper,Chizu Shimokawa,Ninian Blackburn

doi:10.1038/s42003-018-0082-y

Abstract

The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors. Peptidylglycine α-hydroxylating monooxygenase (PHM) is a dicopper enzyme that catalyzes hydroxylation of the α-carbon of glycine-extended peptides for the formation of des-glycine amidated peptides. Here, we present the structures of apo-PHM and of mutants of one of the copper sites (H107A, H108A, and H172A) determined in the presence and absence of citrate. Together, these structures show that the absence of one copper changes the conformational landscape of PHM. In one of these structures, a large interdomain rearrangement brings residues from both copper sites to coordinate a single copper (closed conformation) indicating that full copper occupancy is necessary for locking the catalytically competent conformation (open). These data suggest that in addition to their required participation in catalysis, the redox-active metals play an important structural role.

Highlights

The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors
The structure is strikingly similar to the wild-type holoenzyme (PDB ID: 1PHM) (RMSD 1.6 Å for 1214 main chain atoms), with the largest differences restricted to the loops connecting the β-strands (Supplementary Figure 1A)
Peptidylglycine α-hydroxylating monooxygenase (PHM) is an example of a copper enzyme that utilizes highly evolved reaction chemistry to catalyze a difficult hydroxylation believed to proceed via cupric-superoxomediated radical chemistry

Summary

Introduction

The structures of metalloproteins that use redox-active metals for catalysis are usually exquisitely folded in a way that they are prearranged to accept their metal cofactors. In one of these structures, a large interdomain rearrangement brings residues from both copper sites to coordinate a single copper (closed conformation) indicating that full copper occupancy is necessary for locking the catalytically competent conformation (open) These data suggest that in addition to their required participation in catalysis, the redox-active metals play an important structural role. PAL, a Zn-containing enzyme, completes the reaction, yielding the αamidated peptide product plus glyoxylate Both domains have broad substrate specificity: peptides with all 20 amino acid amides have been isolated[3]. The two copper atoms in PHM, CuH, and CuM (located in the N- and Csubdomains, respectively), each use a single reducing equivalent from ascorbate to catalyze the reduction of molecular oxygen for the hydroxylation of the Cα of glycine at the carboxy-terminus of the peptide substrate[37,38]. The catalytic chemistry must proceed within the confines of a complex cellular transport machinery, with the overall functioning of the system needed to balance the requirements of selective metalation with the structural determinants of catalytic function

Methods

Results

Conclusion