Substrate selectivities of proline hydroxylases

Shibasaki, Takeshi; Sakurai, Wataru; Hasegawa, Asako; Uosaki, Youichi; Mori, Hideo; Yoshida, Mayumi; Ozaki, Akio

doi:10.1016/s0040-4039(99)00944-2

Cited by 53 publications

(32 citation statements)

References 15 publications

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“…Although both FoPip4H and AnPip4H had catalytic properties similar to those of bacterial proline hydroxylases with respect to kinetic parameters and optimal reaction conditions (14,16), their amino acid sequences showed no homology to those of known bacterial proline hydroxylases. More importantly, FoPip4H and AnPip4H preferentially reacted with the substrate L-Pip rather than L-Pro, in contrast to bacterial proline hydroxylases, which favor L-Pro over L-Pip (17,28). Furthermore, we found that FoPip4H is an inducible enzyme strictly regulated by L-Pip but not by L-Pro (Fig.…”

Section: Figmentioning

confidence: 67%

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: Figmentioning

confidence: 67%

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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“…Kuhlmann and Bremer (14) have previously noted that the ectABC gene cluster in Streptomyces coelicolor A3 (2) (37) is immediately followed by a gene that encodes a protein with limited sequence identity (27%) to a L-proline 4-hydroxylase from Dactylosporangium sp (38). This observation suggested to us that the function of this gene product from S. coelicolor A3 (2) might have been incorrectly annotated during the genome sequencing project and may actually serve as an ectoine hydroxylase.…”

Section: Dsm Number Medium (Nacl Molarity) Glutamate Proline Ectoine mentioning

confidence: 99%

“…strain RH1 (38), a L-proline 3-hydroxylase from the Streptomyces sp. strain TH1 (39), and a L-proline 4-hydroxylase from the Streptomyces griseoviridus strain P8648 (40).…”

Section: Dsm Number Medium (Nacl Molarity) Glutamate Proline Ectoine mentioning

confidence: 99%

“…The various EctD proteins exhibit a limited amino acid sequence identity (26% to 32%) to the L-proline 4-hydroxylase from Dactylosporangium sp. strain RH1 (38). This latter protein is a member of the non-heme-containing, iron(II)-and 2-oxoglutarate-dependent dioxygenase superfamily (EC 1.14.11.2) (41-44), members of which contain a highly conserved iron-binding motif that is referred to in the literature as the 2-His-1-carboxylate facial triad (44).…”

Section: Journal Of Biological Chemistry 31151mentioning

confidence: 99%

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Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase

Bursy

Pierik

Pica

et al. 2007

Journal of Biological Chemistry

146

258

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By using natural abundance 13 C NMR spectroscopy, we investigated the types of compatible solutes synthesized in a variety of Bacilli under high salinity growth conditions. Glutamate, proline, and ectoine were the dominant compatible solutes synthesized by the various Bacillus species. The majority of the inspected Bacilli produced the tetrahydropyrimidine ectoine in response to high salinity stress, and a subset of these also synthesized a hydroxylation derivative of ectoine, 5-hydroxyectoine. In Salibacillus salexigens, a representative of the ectoine-and 5-hydroxyectoine-producing species, ectoine production was linearly correlated with the salinity of the growth medium and dependent on an ectABC biosynthetic operon. The formation of 5-hydroxyectoine was primarily a stationary growth phase phenomenon. The enzyme responsible for ectoine hydroxylation (EctD) was purified from S. salexigens to apparent homogeneity. The EctD protein was shown in vitro to directly hydroxylate ectoine in a reaction dependent on iron(II), molecular oxygen, and 2-oxoglutarate. We identified the structural gene (ectD) for the ectoine hydroxylase in S. salexigens. Northern blot analysis showed that the transcript levels of the ectABC and ectD genes increased as a function of salinity. Many EctDrelated proteins can be found in data base searches in various Bacteria. Each of these bacterial species also contains an ect-ABC ectoine biosynthetic gene cluster, suggesting that 5-hydroxyectoine biosynthesis strictly depends on the prior synthesis of ectoine. Our data base searches and the biochemical characterization of the EctD protein from S. salexigens suggest that the EctD-related ectoine hydroxylases are members of a new subfamily within the non-heme-containing, iron(II)-and 2-oxoglutarate-dependent dioxygenase superfamily (EC 1.14.11).

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“…Sequence analysis of the chromosomal region for hypophosphite oxidation revealed five open reading frames, htxABCDE, which appear to form a transcriptional unit. Analysis of the predicted amino acid sequence of the htxA gene product indicates that it is most similar to members of the 2-oxoglutarate-dependent dioxygenase family, having 26% identity to proline 4-hydroxylase from Dactylosporangium (13). The putative htxBCDE products likely comprise a binding protein-dependent hypophosphite transporter.…”

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confidence: 99%

Isolation and Biochemical Characterization of Hypophosphite/ 2-Oxoglutarate Dioxygenase

White

Metcalf

2002

Journal of Biological Chemistry

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The htxA gene is required for the oxidation of hypophosphite in Pseudomonas stutzeri WM88 (Metcalf, W. W., and Wolfe, R. S. (1998) J. Bacteriol. 180, 5547-5558). Amino acid sequence comparisons suggest that hypophosphite:2-oxoglutarate dioxygenase (HtxA) is a novel member of the 2-oxoglutarate-dependent dioxygenase enzyme family. To provide experimental support for this hypothesis, HtxA was overproduced in Escherichia coli and purified to apparent homogeneity. Recombinant HtxA is identical to the native enzyme based on amino terminus sequencing and mass spectral analysis, and it catalyzes the oxidation of hypophosphite to phosphite in a process strictly dependent on 2-oxoglutarate, ferrous ions, and oxygen. Succinate and phosphite are stoichiometrically produced, indicating a strict coupling of the reaction. Size exclusion analysis suggests that HtxA is active as a homodimer, and maximal activity is observed at pH 7.0 and at 27°C. The apparent K m values for hypophosphite and 2-oxoglutarate were 0.58 ؎ 0.04 mM and 10.6 ؎ 1.4 M, respectively. V max and k cat values were determined to be 10.9 ؎ 0.30 mol min ؊1 mg ؊1 and 355 min ؊1 , respectively. 2-Oxoadipate and pyruvate substitute poorly for 2-oxoglutarate as a cosubstrate. The highest specific activity is observed with hypophosphite as substrate, but HtxA is also able to oxidize formate and arsenite at significant rates. The substrate analog inhibitors, formate and nitrate, significantly reduce HtxA activity.The current view of phosphorus metabolism dictates that, unlike other elements essential for growth, phosphorus does not undergo a biologically catalyzed oxidation-reduction cycle in nature. The biochemistry involving this essential and often limiting nutrient is typically thought to be restricted to the formation and hydrolysis of phosphate esters, in which phosphorus exists in its most oxidized state (P 5ϩ ). However, the number of microorganisms capable of using reduced phosphorus compounds as the sole source of phosphorus or able to synthesize reduced phosphorus compounds clearly demonstrates that this is not the case (1-5). Although microbial metabolism of reduced phosphorus compounds has been documented in the literature for several decades, it has remained unexplored in any detail on either the biochemical or genetic levels until recently. This is especially true with respect to the microbial oxidation of the reduced P i compounds, phosphite (P 3ϩ ) and hypophosphite (P ϩ ). Microbial growth on hypophosphite or phosphite as the sole source of phosphorus has been reported in such microorganism as Escherichia coli, Bacillus spp., Pseudomonas fluorescens, Klebsiella aerogenes, and Erwinia spp. (2, 4, 6 -9); however, little is known about the biochemistry of hypophosphite or phosphite oxidation in these organisms. In P. fluorescens, activity of partially purified phosphorus-oxidizing enzyme was demonstrated to be NAD ϩ -dependent and specific for phosphite; however, a more detailed analysis was not completed (10). Similarly, hypophosphite oxida...

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Substrate selectivities of proline hydroxylases

Cited by 53 publications

References 15 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

Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase

Isolation and Biochemical Characterization of Hypophosphite/ 2-Oxoglutarate Dioxygenase

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