Crystal Structure of the Ectoine Hydroxylase, a Snapshot of the Active Site

Höppner, Astrid; Widderich, Nils; Lenders, Michael H. H.; Bremer, Erhard; Smits, Sander H. J.

doi:10.1074/jbc.m114.576769

Cited by 34 publications

(93 citation statements)

References 68 publications

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“…B), and such a gene was also not present anywhere else in their genome. The ectoine hydroxylase (EctD) is an oxygen‐dependent enzyme (Bursy et al ., ; Höppner et al ., ; Widderich et al ., ). Since members of the Methanothrix and Methanobacterium genera are all strict anaerobes, the absence of ectD in the genomes of these methanogens is readily understandable.…”

Section: Resultsmentioning

confidence: 99%

“…). Such a quaternary assembly has previously also been reported for ectoine synthases (Ono et al ., ; Kobus et al ., ) and ectoine hydroxylases (Höppner et al ., ; Widderich et al ., ) from members of the Bacteria .…”

Section: Resultsmentioning

confidence: 99%

“…A). A considerable number of ectoine producers also synthesize an ectoine derivative, 5‐hydroxyectoine, through a stereospecific reaction that is catalysed by the ectoine hydroxylase (EctD) (EC 1.14.11) (Bursy et al ., ; Höppner et al ., ; Widderich et al ., 2014a,b) (Fig. A).…”

Section: Introductionmentioning

confidence: 99%

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Strangers in the archaeal world: osmostress‐responsive biosynthesis of ectoine and hydroxyectoine by the marine thaumarchaeon Nitrosopumilus maritimus

Widderich

Czech

Elling

et al. 2016

Environmental Microbiology

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139

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Ectoine and hydroxyectoine are compatible solutes widely synthesized by members of the Bacteria to cope with high osmolarity surroundings. Inspection of 557 archaeal genomes revealed that only 12 strains affiliated with the Nitrosopumilus, Methanothrix or Methanobacterium genera harbour ectoine/hydroxyectoine gene clusters. Phylogenetic considerations suggest that these Archaea have acquired these genes through horizontal gene transfer events. Using the Thaumarchaeon 'Candidatus Nitrosopumilus maritimus' as an example, we demonstrate that the transcription of its ectABCD genes is osmotically induced and functional since it leads to the production of both ectoine and hydroxyectoine. The ectoine synthase and the ectoine hydroxylase were biochemically characterized, and their properties resemble those of their counterparts from Bacteria. Transcriptional analysis of osmotically stressed 'Ca. N. maritimus' cells demonstrated that they possess an ectoine/hydroxyectoine gene cluster (hyp-ectABCD-mscS) different from those recognized previously since it contains a gene for an MscS-type mechanosensitive channel. Complementation experiments with an Escherichia coli mutant lacking all known mechanosensitive channel proteins demonstrated that the (Nm)MscS protein is functional. Hence, 'Ca. N. maritimus' cells cope with high salinity not only through enhanced synthesis of osmostress-protective ectoines but they already prepare themselves simultaneously for an eventually occurring osmotic down-shock by enhancing the production of a safety-valve (NmMscS).

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Strangers in the archaeal world: osmostress‐responsive biosynthesis of ectoine and hydroxyectoine by the marine thaumarchaeon Nitrosopumilus maritimus

Widderich

Czech

Elling

et al. 2016

Environmental Microbiology

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139

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“…2B) in complex with α-KG and mononuclear iron (Table S3). The central core of both proteins comprises 11 β-strands containing a jelly-roll or β-sandwich fold, characteristic for the α-KG-dependent oxygenase family (21,22), and the proteins show similarity to known non-heme iron dioxygenases (23,24). The β-barrel fold is flanked by four α-helices and several 3 10 helices.…”

Section: Resultsmentioning

confidence: 99%

“…Diffraction data were collected at the European Synchrotron Radiation Facility (ESRF), processed using XDS (36) or MOSFLM (37), and scaled with SCALA or AIMLESS from the CCP4 suite (38). The structure of SnoN was determined by molecular replacement with Phaser (39) using an ensemble of PDB entries 2OPW, 2A1X (23), and 2FCT (40), which show about 20-24% sequence identity, as template. The model obtained was refined using Arp/warp (41), COOT (42), Phenix (43), and REFMAC5 (44).…”

Section: Methodsmentioning

confidence: 99%

Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway

Siitonen

Selvaraj

Niiranen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Nogalamycin, an aromatic polyketide displaying high cytotoxicity, has a unique structure, with one of the carbohydrate units covalently attached to the aglycone via an additional carbon-carbon bond. The underlying chemistry, which implies a particularly challenging reaction requiring activation of an aliphatic carbon atom, has remained enigmatic. Here, we show that the unusual C5′′-C2 carbocyclization is catalyzed by the non-heme iron α-ketoglutarate (α-KG)-dependent SnoK in the biosynthesis of the anthracycline nogalamycin. The data are consistent with a mechanistic proposal whereby the Fe(IV) = O center abstracts the H5′′ atom from the amino sugar of the substrate, with subsequent attack of the aromatic C2 carbon on the radical center. We further show that, in the same metabolic pathway, the homologous SnoN (38% sequence identity) catalyzes an epimerization step at the adjacent C4′′ carbon, most likely via a radical mechanism involving the Fe(IV) = O center. SnoK and SnoN have surprisingly similar active site architectures considering the markedly different chemistries catalyzed by the enzymes. Structural studies reveal that the differences are achieved by minor changes in the alignment of the substrates in front of the reactive ferryl-oxo species. Our findings significantly expand the repertoire of reactions reported for this important protein family and provide an illustrative example of enzyme evolution.natural product biosynthesis | Streptomyces | crystal structure | α-ketoglutarate-dependent oxygenase | iron-dependent oxygenase

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Management of Osmotic Stress by Bacillus Subtilis : Genetics and Physiology

Hoffmann

Bremer

2016

Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria

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Crystal Structure of the Ectoine Hydroxylase, a Snapshot of the Active Site

Cited by 34 publications

References 68 publications

Strangers in the archaeal world: osmostress‐responsive biosynthesis of ectoine and hydroxyectoine by the marine thaumarchaeon Nitrosopumilus maritimus

Strangers in the archaeal world: osmostress‐responsive biosynthesis of ectoine and hydroxyectoine by the marine thaumarchaeon Nitrosopumilus maritimus

Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway

Management of Osmotic Stress by Bacillus Subtilis : Genetics and Physiology

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