Pipecolic Acid Hydroxylases: A Monophyletic Clade among <i>cis</i>‐Selective Bacterial Proline Hydroxylases that Discriminates <scp>l</scp>‐Proline

Mattay, Johanna; Hüttel, Wolfgang

doi:10.1002/cbic.201700187

Cited by 34 publications

(35 citation statements)

References 28 publications

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“…Through a comparison of retention times, the cis-diastereomers of 3-or 4-hydroxylated 4-Me-Pro (4-Me-cis-3-Hyp and 4-Me-cis-4-Hyp, respectively) could be excluded as putative products. References of these compounds were readily available by hydroxylation of 4-Me-Pro with cis-3-PH type II and the pipecolic acid hydroxylase GetF (25). We therefore speculate that the novel product is trans-4-hydroxymethyl-L-proline (4-HyMe-Pro), formed through methyl group hydroxylation in analogy to the generation of 4-HyMe-3-Hyp (Fig.…”

Section: Resultsmentioning

confidence: 98%

Cryptic Production of trans -3-Hydroxyproline in Echinocandin B Biosynthesis

Mattay

Houwaart

Hüttel

2018

Appl Environ Microbiol

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Echinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-l-prolines: -4-hydroxy-l-proline and, in most echinocandin structures, (-2,3)-3-hydroxy-(-2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis by , both amino acids are found in pneumocandin A, while in pneumocandin B the latter residue is replaced by -3-hydroxy-l-proline (3-Hyp). We have recently reported that all three amino acids are generated by the 2-oxoglutarate-dependent proline hydroxylase GloF. In echinocandin B biosynthesis by species, 3-Hyp derivatives have not been reported. Here we describe the heterologous production and kinetic characterization of HtyE, the 2-oxoglutarate-dependent proline hydroxylase from the echinocandin B biosynthetic cluster in Surprisingly, l-proline hydroxylation with HtyE resulted in an even higher proportion (∼30%) of 3-Hyp than that with GloF. This suggests that the selectivity for methylated 3-Hyp in echinocandin B biosynthesis is due solely to a substrate-specific adenylation domain of the nonribosomal peptide synthetase. Moreover, we observed that one product of HtyE catalysis, 3-hydroxy-4-methyl-l-proline, is slowly further oxidized at the methyl group, giving 3-hydroxy-4-hydroxymethyl-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced by Secondary metabolites from bacteria and fungi are often produced by sets of biosynthetic enzymes encoded in distinct gene clusters. Usually, each enzyme catalyzes one biosynthetic step, but multiple reactions are also possible. Pneumocandins A and B are produced by the fungus They belong to the echinocandin family, a group of nonribosomal cyclic lipopeptides that exhibit a strong antifungal activity. Chemical derivatives are important drugs for the treatment of systemic fungal infections. We have recently shown that in the biosynthesis of pneumocandins A and B, three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis in produces the same hydroxyprolines, with an increased proportion of-3-hydroxyproline. However, echinocandin B biosynthesis does not require -3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation in and provides an unusual insight into peptide antibiotic biosynthesis-namely, the complex interplay between the selectivity of a hydroxylase and the substrate specificity of a nonribosomal peptide synthetase.

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Section: Resultsmentioning

confidence: 98%

Cryptic Production of trans -3-Hydroxyproline in Echinocandin B Biosynthesis

Mattay

Houwaart

Hüttel

2018

Appl Environ Microbiol

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“…Interestingly, the fungal enzymes GetF and PiFa preferentially accept pipecolic acid, whereas L-proline is converted only to a very small extent. Using site-directed mutagenesis, the group of Hüttel attempted to shift enzyme activity towards L-proline, albeit with little success [95].…”

Section: L-proline Hydroxylasesmentioning

confidence: 99%

Industrial Application of 2-Oxoglutarate-Dependent Oxygenases

Peters

Buller

2019

Catalysts

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C–H functionalization is a chemically challenging but highly desirable transformation. 2-oxoglutarate-dependent oxygenases (2OGXs) are remarkably versatile biocatalysts for the activation of C–H bonds. In nature, they have been shown to accept both small and large molecules carrying out a plethora of reactions, including hydroxylations, demethylations, ring formations, rearrangements, desaturations, and halogenations, making them promising candidates for industrial manufacture. In this review, we describe the current status of 2OGX use in biocatalytic applications concentrating on 2OGX-catalyzed oxyfunctionalization of amino acids and synthesis of antibiotics. Looking forward, continued bioinformatic sourcing will help identify additional, practical useful members of this intriguing enzyme family, while enzyme engineering will pave the way to enhance 2OGX reactivity for non-native substrates.

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“…In addition, the biosynthetic gene cluster of 1 contains several genes encoding tailoring enzymes, including two iron‐ and α‐ketoglutarate‐dependent enzymes (Fe/αKGs), GetF and GetI (Figure B). GetF was recently characterized as an l ‐pipecolic acid hydroxylase responsible for the production of the 3‐hydroxy‐ l ‐pipecolic acid monomer ( 2 ) . GetI was initially proposed to catalyze the β‐hydroxylation of 2‐chloro‐ l ‐histidine, either as the free or peptidyl carrier protein (PCP)‐bound amino acid ( 3 ) .…”

Section: Figurementioning

confidence: 99%

Characterization of a Citrulline 4‐Hydroxylase from Nonribosomal Peptide GE81112 Biosynthesis and Engineering of Its Substrate Specificity for the Chemoenzymatic Synthesis of Enduracididine

2019

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The GE81112 tetrapeptides are a small family of unusual nonribosomal peptide congeners with potent inhibitory activity against prokaryotic translation initiation. With the exception of the 3‐hydroxy‐l‐pipecolic acid unit, little is known about the biosynthetic origins of the non‐proteinogenic amino acid monomers of the natural product family. Here, we elucidate the biogenesis of the 4‐hydroxy‐l‐citrulline unit and establish the role of an iron‐ and α‐ketoglutarate‐dependent enzyme (Fe/αKG) in the pathway. Homology modelling and sequence alignment analysis further facilitate the rational engineering of this enzyme to become a specific 4‐arginine hydroxylase. We subsequently demonstrate the utility of this engineered enzyme in the synthesis of a dipeptide fragment of the antibiotic enduracidin. This work highlights the value of applying a bioinformatics‐guided approach in the discovery of novel enzymes and engineering of new catalytic activity into existing ones.

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Pipecolic Acid Hydroxylases: A Monophyletic Clade among cis‐Selective Bacterial Proline Hydroxylases that Discriminates l‐Proline

Cited by 34 publications

References 28 publications

Cryptic Production of trans -3-Hydroxyproline in Echinocandin B Biosynthesis

Cryptic Production of trans -3-Hydroxyproline in Echinocandin B Biosynthesis

Industrial Application of 2-Oxoglutarate-Dependent Oxygenases

Characterization of a Citrulline 4‐Hydroxylase from Nonribosomal Peptide GE81112 Biosynthesis and Engineering of Its Substrate Specificity for the Chemoenzymatic Synthesis of Enduracididine

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