New insights into copper monooxygenases and peptide amidation: structure, mechanism and function

  title={New insights into copper monooxygenases and peptide amidation: structure, mechanism and function
  author={Sean T. Prigge and Richard E. Mains and Betty A. Eipper and L. Mario Amzel},
  journal={Cellular and Molecular Life Sciences CMLS},
Abstract. Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-Cα bond. In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine α-amidating monooxygenase (PAM). The PAM gene encodes… 

Peptidylglycine α‐Hydroxylating Monooxygenase (PHM)

The structure and function of PHM have been broadly studied, the pathway of its electron transfer, the nature of the metal-oxygen species, and details of the mechanism are still being investigated.

Peptidyl‐α‐Hydroxyglycine α‐Amidating Lyase (PAL)

The X-ray crystal structure of the PAL catalytic core (PALcc), alone and in complex with the nonpeptidic substrate α-hydroxyhippuric acid, shows that PAL folds as a six-bladed β-propeller.

Evidence for substrate preorganization in the peptidylglycine α-amidating monooxygenase reaction describing the contribution of ground state structure to hydrogen tunneling.

The authors' data show that all Ν-acylglycines bind sequentially to PHM in an equilibrium-ordered fashion, and the decrease in KIE with hydrophobicity was attributed to a preorganization event which decreased reorganization energy by decreasing the conformational sampling associated with ground state substrate binding.

Reaction Mechanism of the Bicopper Enzyme Peptidylglycine α-Hydroxylating Monooxygenase*

A computational study proposes a new mechanism for the reaction mechanism of Peptidylglycine α-hydroxylating monooxygenase, suggesting that the most likely abstracting species is [CuOOH]2+.

Coordination of peroxide to the CuM center of peptidylglycine α-hydroxylating monooxygenase (PHM): structural and computational study

The structure of the oxidized form of PHM complexed with hydrogen peroxide is determined and the geometry of the observed side-on coordinated peroxide ligand in L3CuM(II)O22− is in good agreement with the results of a hybrid quantum mechanical–molecular mechanical optimization of this species.

Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations

The first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo is provided, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates.

The catalytic copper of peptidylglycine alpha-hydroxylating monooxygenase also plays a critical structural role.

Structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role, in the Peptidylglycine alpha-hydroxylating monooxygenase mutant.

HHM motif at the CuH-site of peptidylglycine monooxygenase is a pH-dependent conformational switch.

The results establish that M109 is indeed the coordinating ligand and confirm the prediction that the low pH structural transition with associated loss of activity is abrogated when the M109 thioether is absent, suggesting a more general utility for the HHM motif as a copper- and pH-dependent conformational switch.

Peptide amidation: Production of peptide hormonesin vivo andin vitro

This review presents the current situation regarding amidation, with a special emphasis on the industrial production of peptide hormones, as well as various strategies utilizing PAM, carboxypeptidase-Y enzymes, and chemical synthesis for producing peptide amidesin vitro.



Novel substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase.

It is reported that PAM readily catalyzes three alternate monooxygenase reactions--sulfoxidation, amine N-dealkylation, and O-deALKylation--and that the olefinic substrate analogues trans-benzoylacrylic acid and 4-phenyl-3-butenoic acid are potent time-dependent inactivators of PAM, with inactivation exhibiting the characteristics expected for mechanism-based inhibition.

A molluscan peptide α‐amidating enzyme precursor that generates five distinct enzymes

The cellular expression of the LPAM gene is restricted to neurons that synthesize amidated peptides, which underscores the critical importance of regulation of peptide α‐amidation.

Pyruvate-extended Amino Acid Derivatives as Highly Potent Inhibitors of Carboxyl-terminal Peptide Amidation*

The first potent inhibitors for peptidylamidoglycolate lyase are introduced, which can be viewed as pyruvate-extended N-acetyl amino acids, which are highly potent, pure competitive inhibitors of PGL.

Structural and functional investigations on the role of zinc in bifunctional rat peptidylglycine alpha-amidating enzyme.

The structure and coordination of the zinc center has been investigated by X-ray absorption spectroscopy and it is shown that alpha-AE requires 1.1 +/- 0.2 mol of zinc/mol of enzyme for maximal (S)-N-dansyl-Tyr-Val-alpha-hydroxyglycine dealkylation activity.

Amidation of bioactive peptides: the structure of peptidylglycine alpha-hydroxylating monooxygenase.

These structures strongly suggest that the PHM reaction proceeds via activation of substrate by a copper-bound oxygen species, and can be directly extended to dopamine beta-monooxygenase.

Kinetic and inhibition studies on substrate channelling in the bifunctional enzyme catalysing C-terminal amidation.

The results demonstrate the kinetic independence of the mono-oxygenase and lyase domains, and provide no evidence for substrate channelling between these domains in the bifunctional amidating enzyme.

Molecular Cloning of a Peptidylglycine α-Hydroxylating Monooxygenase from Sea Anemones

Using PCR and degenerated oligonucleotides derived from conserved regions of PHM, a PHM is cloned from the sea anemone Calliactis parasitica showing 42% amino acid sequence identity with rat PHM.

Inhibition of peptidylglycine alpha-amidating monooxygenase by N-substituted homocysteine analogs.

Dipeptides containing a C-terminal homocysteine and an N-acylated hydrophobic amino acid were found to inhibit PAM with IC50s in the low nanomolar range, indicating that the compounds have sufficient potency and intracellular bioavailability to aid future studies focused on neuropeptide function and the contributions of neuropePTides to various disease processes.