Role for an Essential Tyrosine in Peptide Amidation

De, Mithu; Bell, Joseph; Blackburn, Ninian J.; Mains, Richard E.; Eipper, Betty A.

doi:10.1074/jbc.m513886200

Cited by 34 publications

(45 citation statements)

References 68 publications

(97 reference statements)

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“…Monofunctional SmPAL is a soluble, secreted enzyme, similar in sequence to other PALs such as the secreted Drosophila enzyme, dPAL2 [35], retaining four conserved cysteines for the maintenance of secondary structure, plus four cysteine residues unique to SmPAL, and a tyrosine residue known to be essential for functionality [39, 40]. Yet there are key amino acid substitutions in SmPAL, in the Ca 2+ binding site, and where the amino acid preceding the α-hydroxyglycine and the PAL active site interact, which set it apart from the host enzyme.…”

Section: Discussionmentioning

confidence: 99%

A PAL for Schistosoma mansoni PHM

Atkinson¹,

McVeigh²,

Kimber³

et al. 2010

Molecular and Biochemical Parasitology

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Parasitic helminth neuromuscular function is a proven target for chemotherapeutic control. Although neuropeptide signaling plays a key role in helminth motor function, it has not yet provided targets for known anthelmintics. The majority of biologically active neuropeptides display a C-terminal amide (NH 2 ) motif, generated exclusively by the sequential action of two enzymes, peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidylglycine α-amidating lyase (PAL). Further to our previous description of a monofunctional PHM enzyme (SmPHM) from the human blood fluke Schistosoma mansoni, here we describe a cDNA encoding S. mansoni PAL (SmPAL). SmPAL is a monofunctional enzyme which, following heterologous expression, we find to have functionally similar catalytic activity and optimal pH values, but key catalytic core amino acid substitutions, when compared to other known PALs including those found in humans. We have used in situ hybridization to demonstrate that in adult schistosomes, SmPAL mRNA (Sm-pal-1) is expressed in neuronal cell bodies of the central nervous system, consistent with a role for amidated neuropeptides in S. mansoni neuromuscular function. In order to validate SmPAL as a putative drug target we applied published RNA interference (RNAi) methods in efforts to trigger knockdown of Sm-pal-1 transcript in larval schistosomula. Although transcript knock-down was recorded on several occasions, silencing was variable and inconsistent and did not associate with any observable aberrant phenotype. The inconsistent outcomes of RNAi suggest that there may be tissue-specific differences in the applicability of RNAi methods for S. mansoni, with neuronal targets proving more difficult or refractory to knockdown. The key role played by schistosome amidating enzymes in neuropeptide maturation make them appealing as drug targets; their validation as such will depend on the development of more robust reverse genetic tools to facilitate efficient neuronal gene function studies.

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Section: Discussionmentioning

confidence: 99%

A PAL for Schistosoma mansoni PHM

Atkinson¹,

McVeigh²,

Kimber³

et al. 2010

Molecular and Biochemical Parasitology

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“…The PAL domain, unique to the PAM system, is a zinc and calcium-dependent and catalyzes the dealkylation of the carbinolamide to the corresponding amide and glyoxylate (Scheme 1a). 13,14…”

Section: Introductionmentioning

confidence: 99%

Imino-oxy Acetic Acid Dealkylation as Evidence for an Inner-Sphere Alcohol Intermediate in the Reaction Catalyzed by Peptidylglycine α-Hydroxylating Monooxygenase

2009

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Peptidylglycine α-hydroxylating monooxygenase (PHM, EC 1.14.17.3) catalyzes the stereospecific hydroxylation of a glycyl α-carbon in a reaction that requires O2 and ascorbate. Subsequent dealkylation of the α-hydroxyglycine by another enzyme, peptidylamidoglycolate lyase (PAL. EC 4.3.2.5), yields a bioactive amide and glyoxylate. PHM is a non-coupled, type II dicopper monooxygenase which activates O2 at only a single copper atom, CuM. In this study, the PHM mechanism was probed using a non-natural substrate, benzaldehyde imino-oxy acetic acid (BIAA). PHM catalyzes the O-oxidative dealkylation of BIAA to benzaldoxime and glyoxylate with no involvement of PAL. The minimal kinetic mechanism for BIAA was shown to be steady-state ordered using primary deuterium kinetic isotope effects. The D(V/K)APPARENT, BIAA decreased from 14.7 ± 1.0 as [O2] → 0 to 1.0 ± 0.2 as [O2] → ∞ suggesting the dissociation rate constant from the PHM·BIAA complex decreases as [O2] increases; thereby, reducing the steady-state concentration of [PHM]free. BIAA was further used to differentiate between potential oxidative Cu/O species using a QM/MM reaction coordinate simulation to determine which species could yield product O-dealkylation that matched our experimental data. The results of this study provided compelling evidence for the presence of a covalently linked CuII-alkoxide intermediate with a quartet spin state responsible BIAA oxidation.

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“…These residues are conserved in the enzymes from C. bullatus and C. geographus . These include the four cysteine residues involved in disulfide formation, (Cys 492, 512, 559 and 570), histidine residues that coordinate a zinc ion at the active site (H444, H547, H641), or have structural roles (H393, H462) and other residues having catalytic roles (Y511, R563, D562, R394), structural roles (D449, D458, and D509) (Attenborough et al, 2012; Chufan et al, 2009; De et al, 2006; Kolhekar et al, 2002). …”

Section: Resultsmentioning

confidence: 99%

Characterization of the peptidylglycine α-amidating monooxygenase (PAM) from the venom ducts of neogastropods, Conus bullatus and Conus geographus

Ul-Hasan

Burgess

Gajewiak

et al. 2013

Toxicon

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Cone snails, genus Conus, are predatory marine snails that use venom to capture their prey. This venom contains a diverse array of peptide toxins, known as conotoxins, which undergo a diverse set of posttranslational modifications. Amidating enzymes modify peptides and proteins containing a C-terminal glycine residue, resulting in loss of the glycine residue and amidation of the preceding residue. A significant fraction of peptides present in the venom of cone snails contain C-terminal amidated residues, which are important for optimizing biological activity. This study describes the characterization of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), present in the venom duct of cone snails, Conus bullatus and Conus geographus. PAM is known to carry out two functions, peptidyl α-hydroxylating monooxygenase (PHM) and peptidylamido-glycolate lyase (PAL). In some animals, such as Drosophila melanogaster, these two functions are present in separate polypeptides, working as individual enzymes. In other animals, such as mammals and in Aplysia californica, PAM activity resides in a single, bifunctional polypeptide. Using specific oligonucleotide primers and reverse transcription-polymerase chain reaction we have identified and cloned from the venom duct cDNA library, a cDNA with 49% homology to PAM from A. californica. We have determined that both the PHM and PAL activities are encoded in one mRNA polynucleotide in both C. bullatus and C. geographus. We have directly demonstrated enzymatic activity catalyzing the conversion of dansyl-YVG-COOH to dansyl-YV-NH2 in cloned cDNA expressed in Drosophila S2 cells.

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Role for an Essential Tyrosine in Peptide Amidation

Cited by 34 publications

References 68 publications

A PAL for Schistosoma mansoni PHM

A PAL for Schistosoma mansoni PHM

Imino-oxy Acetic Acid Dealkylation as Evidence for an Inner-Sphere Alcohol Intermediate in the Reaction Catalyzed by Peptidylglycine α-Hydroxylating Monooxygenase

Characterization of the peptidylglycine α-amidating monooxygenase (PAM) from the venom ducts of neogastropods, Conus bullatus and Conus geographus

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