A novel Fe(II)/α-ketoglutarate-dependent dioxygenase from<i>Burkholderia ambifaria</i>has β-hydroxylating activity of<i>N</i>-succinyl l-leucine

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

doi:10.1111/j.1472-765x.2012.03308.x

Cited by 25 publications

(45 citation statements)

References 29 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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“…Five kinds of substrates-N-succinyl-L-leucine (NSLeu), N-succinyl-L-valine (NSVal), N-succinyl-L-isoleucine (NSIle), N-succinyl-Lphenylalanine (NSPhe) and NSDOPA-were synthesized as described previously [13]. The reaction proceeded at 30°C for 2 h and the reaction mixture was composed of 10 mM substrate, 15 mM a-KG, 0.5 mM FeSO 4 Á7H 2 O, 10 mM L-ascorbate, 50 mM Tris-HCl buffer (pH 8.0), and 1 mg ml À1 purified SadA.…”

Section: Activity Assaymentioning

confidence: 99%

“…In previous studies, we have reported an Fe(II)-and a-ketoglutarate (KG)-dependent dioxygenase from Burkholderia ambifaria AMMD (SadA, 30,664 Da) [13,14]. SadA enantioselectively catalyzes the C3-hydroxylation of not only N-succinyl branched-chain L-amino acids but also N-succinyl aromatic L-amino acids to produce the hydroxy amino acids.…”

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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“…This enzyme is useful as a novel biocatalyst for the ( R )-selective hydroxylation at the C-3 position of N -substituted branched-chain L-amino acids, especially N -succinyl-L-leucine (NSLeu), to produce N -succinyl-(2 S ,3 R )-3-hydroxyleucine (NSHLeu) with >99% stereoselectivity (Fig. 1) [10]. (2 S ,3 R )-3-hydroxyleucine is a promising material for the preparation of certain cyclic depsipeptides which function as platelet aggregation inhibitors and is also a component of the antibiotic lysobactin [11], [12].…”

Section: Introductionmentioning

confidence: 99%

Crystal Structure of a Novel N-Substituted L-Amino Acid Dioxygenase from Burkholderia ambifaria AMMD

et al. 2013

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A novel dioxygenase from Burkholderia ambifaria AMMD (SadA) stereoselectively catalyzes the C3-hydroxylation of N-substituted branched-chain or aromatic L-amino acids, especially N-succinyl-L-leucine, coupled with the conversion of α-ketoglutarate to succinate and CO2. To elucidate the structural basis of the substrate specificity and stereoselective hydroxylation, we determined the crystal structures of the SadA.Zn(II) and SadA.Zn(II).α-KG complexes at 1.77 Å and 1.98 Å resolutions, respectively. SadA adopted a double-stranded β-helix fold at the core of the structure. In addition, an HXD/EXnH motif in the active site coordinated a Zn(II) as a substitute for Fe(II). The α-KG molecule also coordinated Zn(II) in a bidentate manner via its 1-carboxylate and 2-oxo groups. Based on the SadA.Zn(II).α-KG structure and mutation analyses, we constructed substrate-binding models with N-succinyl-L-leucine and N-succinyl-L-phenylalanine, which provided new insight into the substrate specificity. The results will be useful for the rational design of SadA variants aimed at the recognition of various N-succinyl L-amino acids.

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

Cited by 25 publications

References 29 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

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

Crystal Structure of a Novel N-Substituted L-Amino Acid Dioxygenase from Burkholderia ambifaria AMMD

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