l-Leucine 5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase that is useful as a biocatalyst

Hibi, Makoto; Kawashima, Takayuki; Sokolov, Pavel M.; Smirnov, Sergey V.; Kodera, Tomohiro; Sugiyama, Masakazu; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

doi:10.1007/s00253-012-4136-7

Cited by 49 publications

(45 citation statements)

References 25 publications

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“…Moreover, the addition of ferrous iron, ␣KG, and ascorbate to the reaction mixture was required for the maximum activity of the F. oxysporum c8D cell lysate. Since similar observations have been made for other amino acid hydroxylases belonging to the Fe/␣KG-DO superfamily (19)(20)(21), these results suggest that the enzyme responsible for L-Pip-hydroxylating activity belongs to the Fe/␣KG-DO superfamily.…”

Section: Resultssupporting

confidence: 57%

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans -4-Hydroxylation of l -Pipecolic Acid

Hibi

Mori

Miyake

et al. 2016

Appl Environ Microbiol

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Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward L-pipecolic acid (L-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme L-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/␣-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio-and stereoselective hydroxylation of L-Pip, and produces optically pure trans-4-hydroxy-L-pipecolic acid (trans-4-L-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had L-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the L and D forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-L-HyPip was successfully developed. Thus, we report a fungal family of L-Pip hydroxylases and the enzymatic preparation of trans-4-L-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities.H ydroxypipecolic acids (HyPips) are naturally occurring sixmembered heterocyclic hydroxy amino acids that are widely distributed in nature. For instance, 4-hydroxy-, 5-hydroxy-, and 4,5-dihydroxypipecolic acids have been found in various members of the plant family (1-4). Likewise, 5-hydroxypipecolic acid was found to be present in mammalian brain and blood (5). HyPips are also components of some peptide antibiotics, terpenoids, and alkaloids, for example, Thus, the enzymatic hydroxylation of L-Pip was achieved as a side reaction of L-proline hydroxylase. To date, specific L-Piphydroxylating enzymes have not been reported, despite the fact that HyPips are important metabolites found in plants and mammals. In the present study, we identified and characterized L-Pip hydroxylases broadly conserved in some filamentous fungi. We found that the newly discovered L-Pip hydroxylases were particularly suitable biocatalysts for the asymmetric hydroxylation of cyclic amino acids, such as pipecolic acids and prolines. Moreover, the widespread distribution of genes encoding L-Pip hydroxylases indicates some important physiological roles of fungal secondary metabolites related to trans-4-L-HyPip. MATERIALS AND METHODSStrains and culture. Microorganisms isolated from soil and plant samples were cultured in GP medium comprised of 0.15% (wt/vol) KH 2 PO 4 , 0.05% (wt/vol) K 2 HPO 4 , 0.1% (wt/vol) NH 4 Cl, 0.03% (wt/vol) MgSO 4 ·7H 2 O, 0.01% (wt/vol) yeast extract, 0.025% (wt/vol) glucose, and 0.1% (wt/vol) L-Pip (pH 7.0) and grown at 28°C with shaking. Escherichia coli JM109 and Rosetta2(DE3) (Novagen, WI, USA) were used as host strains for the overexpression of the recombinant enzymes and cultured at 28°C in LB medium, comprised of 1% (wt/v...

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Section: Resultssupporting

confidence: 57%

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans -4-Hydroxylation of l -Pipecolic Acid

Hibi

Mori

Miyake

et al. 2016

Appl Environ Microbiol

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“…Previously, we reported two Fe(II)/αKG‐dependent dioxygenases, IDO of Bacillus thuringiensis (Hibi et al. 2011) and LdoA of Nostoc punctiforme (Hibi et al. 2012), catalysing different hydroxylating reactions of l‐leucine.…”

Section: Resultsmentioning

confidence: 99%

“…SadA has no apparent homology with other Fe(II) ⁄ aKGdependent dioxygenases known to hydroxylate free amino acids such as L-proline, L-arginine, L-asparagine, L-isoleucine and L-leucine (Mori et al 1997;Shibasaki et al 1999;Yin and Zabriskie 2004;Strieker et al 2007;Kodera et al 2009;Hibi et al 2012). According to the results of Blast searches, 7-deoxy-cylindrospermopsin hydroxylase, CyrI, of Oscillatoria sp.…”

Section: Resultsmentioning

confidence: 99%

A novel Fe(II)/α-ketoglutarate-dependent dioxygenase fromBurkholderia ambifariahas β-hydroxylating activity ofN-succinyl l-leucine

Hibi

Kawashima

Kasahara

et al. 2012

Letters in Applied Microbiology

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An Fe(II)/α‐ketoglutarate‐dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N‐substituted l‐amino acids, which was especially strong with N‐succinyl l‐leucine. With the NMR and LC‐MS analysis, SadA converted N‐succinyl l‐leucine into N‐succinyl l‐threo‐β‐hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing β‐hydroxylation of aliphatic amino acid‐related substances and a potent biocatalyst for the preparation of optically active β‐hydroxy amino acids.

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“…Dioxygenases are involved in the biological processes ranging from antibiotic biosynthesis to oxygen sensing in humans [10][11][12]. The enzymes catalyze a number of oxidation reactions.…”

Section: Introductionmentioning

confidence: 99%

Structural optimization of SadA, an Fe(II)- and α-ketoglutarate-dependent dioxygenase targeting biocatalytic synthesis of N-succinyl-l-threo-3,4-dimethoxyphenylserine

Qin

Miyakawa

Nakamura

et al. 2014

Biochemical and Biophysical Research Communications

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l-Leucine 5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase that is useful as a biocatalyst

Cited by 49 publications

References 25 publications

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans -4-Hydroxylation of l -Pipecolic Acid

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans -4-Hydroxylation of l -Pipecolic Acid

A novel Fe(II)/α-ketoglutarate-dependent dioxygenase fromBurkholderia ambifariahas β-hydroxylating activity ofN-succinyl l-leucine

Structural optimization of SadA, an Fe(II)- and α-ketoglutarate-dependent dioxygenase targeting biocatalytic synthesis of N-succinyl-l-threo-3,4-dimethoxyphenylserine

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