Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli

Handa, Sumit; Spradling, Tyler J.; Dempsey, Daniel R.; Merkler, David J.

doi:10.1016/j.pep.2012.04.012

Cited by 8 publications

(3 citation statements)

References 38 publications

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“…Despite many attempts to express fully active PAM in bacterial, yeast and insect systems, mammalian cell lines remain the system of choice. PHMcc has been expressed in E. coli with an Nterminal fusion to thioredoxin, but with a yield of only 100 μg/litre of E. coli (Handa et al, 2012). Production of recombinant rat PHMcc, PALcc and rat medullary thyroid carcinoma (MTC) PAM in CHO (CHO) cells has yielded >100 mg of pure enzyme (Bauman et al, 2007;Miller et al, 1992), a sufficient quantity to support spectroscopic studies (Blackburn et al, 2000;Eipper et al, 1995;Evans et al, 2006;Jaron & Blackburn, 1999), structural biology (Chufan et al, 2009;Prigge et al, 1997Prigge et al, , 1999 and amidated peptide production at the multi-gram level (Ray et al, 1993).…”

Section: The Phm Reactionmentioning

confidence: 99%

Peptidylglycine α‐amidating monooxygenase as a therapeutic target or biomarker for human diseases

Merkler

Hawley

Eipper

et al. 2022

British J Pharmacology

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Peptides play a key role in controlling many physiological and neurobiological pathways. Many bioactive peptides require a C‐terminal α‐amide for full activity. The bifunctional enzyme catalysing α‐amidation, peptidylglycine α‐amidating monooxygenase (PAM), is the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine functions are dependent upon amidated peptides; additional amidated peptides are growth promoters in tumours. The amidation reaction occurs in two steps, glycine α‐hydroxylation followed by dealkylation to generate the α‐amide product. Currently, most potentially useful inhibitors target the first reaction, which is rate‐limiting. PAM is a membrane‐bound enzyme that visits the cell surface during peptide secretion. PAM is then used again in the biosynthetic pathway, meaning that cell‐impermeable inhibitors or inactivators could have therapeutic value for the treatment of cancer or psychiatric abnormalities. To date, inhibitor design has not fully exploited the structures and mechanistic details of PAM.

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Section: The Phm Reactionmentioning

confidence: 99%

Peptidylglycine α‐amidating monooxygenase as a therapeutic target or biomarker for human diseases

Merkler

Hawley

Eipper

et al. 2022

British J Pharmacology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Despite many attempts to express fully active PAM in bacterial, yeast, and insect systems, mammalian cell lines remain the system of choice. PHMcc has been expressed in E. coli with an Nterminal fusion to thioredoxin, but with a yield of only˜100 μg/liter of E. coli (Handa, Spradling, Dempsey, & Merkler, 2012). Production of recombinant rat PHMcc, PALcc and rat MTC PAM in Chinese hamster ovary (CHO) cells has yielded >100 mg of pure enzyme (Bauman, Ralle, & Blackburn, 2007;Miller et al, 1992), sufficient enzyme to support spectroscopic studies (Blackburn, Rhames, Ralle, & Jaron, 2000;Eipper et al, 1995;Evans, Blackburn, & Klinman, 2006;Jaron & Blackburn, 1999), structural biology (Chufan et al, 2009;Prigge et al, 1997;Prigge, Kolhekar, Eipper, Mains, & Amzel, 1999), and amidated peptide production at the multi-gram level (Ray et al, 1993).…”

Section: πεπτιδε α-αμιδατιον ιν τωο στεπςmentioning

confidence: 99%

Peptidylglycine α-amidating monooxygenase as a therapeutic target or biomarker

Merkler

Hawley

Eipper

et al. 2021

Preprint

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Peptides play a key role in controlling many physiological and neurobiological pathways. Many bioactive peptides require a C-terminal α-amide for full activity. The bifunctional enzyme catalyzing α-amidation, peptidylglycine α-amidating monooxygenase (PAM), is the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine functions are dependent upon amidated peptides; additional amidated peptides are growth promoters in tumors. The amidation reaction occurs in two steps, glycine α-hydroxylation followed by dealkylation to generate the α-amide product. Currently, most potentially useful inhibitors target the first reaction, which is rate-limiting. PAM is a membrane-bound enzyme that visits the cell surface during peptide secretion. PAM is then used again in the biosynthetic pathway, meaning that cell-impermeable inhibitors or inactivators could have therapeutic value for the treatment of cancer or psychiatric abnormalities. To date, inhibitor design has not fully exploited the structures and mechanistic details of PAM.

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“…[229] However, for the production of C-terminal amide peptides, expression of human PAM enzymes were conducted in E. coli. [230] Peptide keto aldehydes generated from split of the glyoxpeptides are also intriguing as peptide keto aldehydes are described to be specific and reversible proteasome inhibitors. [231,232]…”

Section: Monoox Domain Characterisationmentioning

confidence: 99%

Biosynthesis of selected natural products from entomopathogenic bacteria

Westphalen¹

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This work comprises the investigation of four different biosynthesis gene clusters from Xenorhabdus. Xenorhabdus is an entomopathogenic bacterium that lives in mutualistic symbiosis with its Steinernema nematode host and together they infect and kill insect larvae. Xenorhabdus is well known for the production of so-called specialised metabolites and many of these compounds are synthesised by non-ribosomal peptide synthetases (NRPSs) or NRPS-polyketide synthase (PKS)-hybrids. These enzymes are organised in a modular manner and produce structurally very diverse molecules, often with the help of modifying domains and tailoring enzymes. In general, the genes involved in the biosynthesis are organised in so-called biosynthetic gene clusters (BGCs) in the genome of the producing strain. Exchanging the native promoter with an inducible promoter, e.g. PBAD, allows the targeted activation of the BGC and in turn the analysis of the biosynthesis product via LC-MS analysis. The first BGC investigated in this work is responsible for the biosynthesis of xenofuranones. Based on gene deletions, this work shows that the NRPS-like enzyme XfsA produces a carboxylated furanone intermediate which is subsequently decarboxylated by XfsB to yield xenofuranone B. The next step in xenofuranone biosynthesis is the O-methylation of xenofuranone B to yield xenofuranone A. A comparative proteomics approach allowed the identification of four methyltransferase candidates and subsequent gene deletions confirmed one of the candidates to be responsible for methylation of xenofuranone B. The proteome analysis was based on the comparison of X. szentirmaii WT and X. szentirmaii Δhfq because distinct levels of the methylated xenofuranone A were observed when the xfs BGC was activated in either WT or Δhfq strain. Hfq is a global transcriptional regulator whose deletion is associated with the down regulation of natural product biosynthesis in Xenorhabdus. The strong PBAD activation of the xfs BGC also allowed the detection of two novel xenofuranone derivatives which arise from incorporation of one 4-hydroxyphenylpyruvic acid as first or second building block, respectively. PBAD based activation of the second BGC addressed in this work lead to the detection of a novel metabolite and compound purification allowed NMR-based structure elucidation. The molecule exhibits two pyrrolizidine moieties and was named pyrrolizwilline (pyrrolizidine + twin (German: “Zwilling”)). The BGC comprises seven genes and single gene deletions as well as heterologous expression in E. coli and NRPS engineering were conducted to investigate the biosynthesis. The first two genes xhpA and xhpB encode a bimodular NRPS and a monooxygenase which synthesise a pyrrolizixenamide-like structure, similar to PxaA and PxaB in pyrrolizixenamide biosynthesis. It is suggested that the acyl side chain incorporated by XhpA is removed by the α,β-hydrolase XhpG. The keto function is then reduced by two subsequent two electron reductions catalysed by XhpC and XhpD. One of these two reduced pyrrolizidine units most likely is extended with glyoxalate prior to non-enzymatic dimerisation with the second pyrrolizidine moiety. To finally yield pyrrolizwilline, L-valine is incorporated, probably by the free-standing condensation domain XhpF. The third BGC investigated is responsible for the production of a tripeptide composed of β-D-homoserine, α-hydroxyglycine and L-valine and is referred to as glyoxpeptide. This work demonstrates that the previously observed glyoxpeptide derivative is derived from glycerol present in the culture medium. Furthermore, this work shows that the monooxygenase domain, which is found in an unusual position between motifs A8 and A9 within the adenylation domain, is responsible for the α-hydroxylation of glycine. It is suggested that the α-hydroxylation of glycine renders the tripeptide prone to hydrolysis via hemiacetal formation. Hence, the XgsC_MonoOx domain might be an interesting candidate for further NRPS engineering. The fourth BGC addressed is responsible for the production of xildivalines and this work describes two additional derivatives which are detected only when the promoter is exchanged and activated in the X. hominickii WT strain but not in X. hominickii Δhfq. Deletion of the methyltransferase encoding gene xisE results in the production of non-methylated xildivalines. It remains to be determined when the N-methylation of L-valine takes place. It is discussed that the methyltransferase could act on the NRPS released product but also during the assembly. The peptide deformylase is not involved in the proposed biosynthesis as xildivaline production is detected in a ΔxisD strain. The PKS XisB features two adjacent, so-called tandem T domains. The inactivation of the first or the second T domain by point mutation causes decreased production titres of detected xildivalines in the respective mutant strain when compared to the wild type.

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Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli

Cited by 8 publications

References 38 publications

Peptidylglycine α‐amidating monooxygenase as a therapeutic target or biomarker for human diseases

Peptidylglycine α‐amidating monooxygenase as a therapeutic target or biomarker for human diseases

Peptidylglycine α-amidating monooxygenase as a therapeutic target or biomarker

Biosynthesis of selected natural products from entomopathogenic bacteria

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