Carnitine metabolism to trimethylamine by an unusual Rieske-type oxygenase from human microbiota

Dietary intake of L-carnitine can promote cardiovascular diseases in humans through microbial production of trimethylamine (TMA) and its subsequent oxidation to trimethylamine N-oxide (TMAO) by hepatic flavin-containing monooxygenases. Although our microbiota are responsible for TMA formation from carnitine, the underpinning molecular and biochemical mechanisms remain unclear. In this study, using bioinformatics approaches, we first identified a two-component Rieske-type oxygenase/reductase (CntAB) and associated gene cluster proposed to be involved in carnitine metabolism in representative genomes of the human microbiota. CntA belongs to a group of previously uncharacterized Rieske-type proteins and has an unusual "bridging" glutamate but not the aspartate residue, which is believed to facilitate inter-subunit electron transfer between the Rieske centre and the catalytic mononuclear iron centre. Using Acinetobacter baumannii as the model, we then demonstrate that cntAB is essential in carnitine degradation to TMA. Heterologous overexpression of cntAB enables Escherichia coli to produce TMA, confirming that these genes are sufficient in TMA formation. Site-directed mutagenesis experiments have confirmed that this unusual "bridging glutamate" residue in CntA is essential in catalysis and neither mutant (E205D, E205A) is able to produce TMA. Together, our study reveals the molecular and biochemical mechanisms underpinning carnitine metabolism to TMA in human microbiota and assigns the role of this novel group of Rieske-type proteins in microbial carnitine metabolism

Substrate Range and Genetic Analysis of Acinetobacter Vanillate Demethylase

An Acinetobacter sp. genetic screen was used to probe structure-function relationships in vanillate demethylase, a two-component monooxygenase. Mutants with null, leaky, and heat-sensitive phenotypes were isolated. Missense mutations tended to be clustered in specific regions, most of which make known contributions to catalytic activity. The vanillate analogs m-anisate, m-toluate, and 4-hydroxy-3,5-dimethylbenzoate are substrates of the enzyme and weakly inhibit the metabolism of vanillate by wild-type Acinetobacter bacteria. PCR mutagenesis of vanAB, followed by selection for strains unable to metabolize vanillate, yielded mutant organisms in which vanillate metabolism is more strongly inhibited by the vanillate analogs. Thus, the procedure opens for investigation amino acid residues that may contribute to the binding of either vanillate or its chemical analogs to wild-type and mutant vanillate demethylases. Selection of phenotypic revertants following PCR mutagenesis gave an indication of the extent to which amino acid substitutions can be tolerated at specified positions. In some cases, only true reversion to the original amino acid was observed. In other examples, a range of amino acid substitutions was tolerated. In one instance, phenotypic reversion failed to produce a protein with the original wild-type sequence. In this example, constraints favoring certain nucleotide substitutions appear to be imposed at the DNA level

Degradation of BTEX by anaerobic bacteria: physiology and application

Pollution of the environment with aromatic hydrocarbons, such as benzene, toluene, ethylbenzene and xylene (so-called BTEX) is often observed. The cleanup of these toxic compounds has gained much attention in the last decades. In situ bioremediation of aromatic hydrocarbons contaminated soils and groundwater by naturally occurring microorganisms or microorganisms that are introduced is possible. Anaerobic bioremediation is an attractive technology as these compounds are often present in the anoxic zones of the environment. The bottleneck in the application of anaerobic techniques is the lack of knowledge about the anaerobic biodegradation of benzene and the bacteria involved in anaerobic benzene degradation. Here, we review the existing knowledge on the degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria, in particular the physiology and application, including results on the (per)chlorate stimulated degradation of these compounds, which is an interesting new alternative option for bioremediatio

Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP.

Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His10-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120

JMJD5 is a human arginyl C-3 hydroxylase.

Oxygenase-catalysed post-translational modifications of basic protein residues, including lysyl hydroxylations and Nε-methyl lysyl demethylations, have important cellular roles. Jumonji-C (JmjC) domain-containing protein 5 (JMJD5), which genetic studies reveal is essential in animal development, is reported as a histone Nε-methyl lysine demethylase (KDM). Here we report how extensive screening with peptides based on JMJD5 interacting proteins led to the finding that JMJD5 catalyses stereoselective C-3 hydroxylation of arginine residues in sequences from human regulator of chromosome condensation domain-containing protein 1 (RCCD1) and ribosomal protein S6 (RPS6). High-resolution crystallographic analyses reveal overall fold, active site and substrate binding/product release features supporting the assignment of JMJD5 as an arginine hydroxylase rather than a KDM. The results will be useful in the development of selective oxygenase inhibitors for the treatment of cancer and genetic diseases

Differential Expression of the Demosponge (Suberites domuncula) Carotenoid Oxygenases in Response to Light: Protection Mechanism Against the Self-Produced Toxic Protein (Suberitine)

The demosponge Suberites domuncula has been described to contain high levels of a proteinaceous toxin, Suberitine, that displays haemolytic activityIn the present study this 7–8 kDa polypeptide has been isolated and was shown to exhibit also cytotoxic effects on cells of the same species. Addition of retinal, a recently identified metabolite of β-carotene that is abundantly present in S. domuncula was found to reduce both the haemolytic and the cell toxic activity of Suberitine at a molar ratio of 1:1. Spectroscopic analyses revealed that the interaction between β-carotene and Suberitine can be ascribed to a reversible energy transfer reaction. The enzyme that synthesises retinal in the sponge system is the β,β-carotene-15,15′-dioxygenase [carotene dioxygenase]. In order to clarify if this enzyme is the only β-carotene-metabolizing enzyme a further oxygenase had been identified and cloned, the (related) carotenoid oxygenase. In contrast to the dioxygenase, the carotenoid oxygenase could not degrade β-carotene or lycopene in Escherichia coli strains that produced these two carotenoids; therefore it had been termed related-carotenoid oxygenase. Exposure of primmorphs to light of different wavelengths from the visible spectrum resulted after 3 days in a strong upregulation of the dioxygenase in those 3D-cell aggregates that had been incubated with β-carotene. The strongest effect is seen with blue light at a maximum around 490 nm. It is concluded that the toxin Suberitine is non-covalently modified by retinal, the cleavage product from β-carotene via the enzyme carotene dioxygenase, a light inducible oxygenase. Hence, this study highlights that in S. domuncula the bioactive metabolite, retinal, has the property to detoxify its homologous toxin

Whole-cell redox biocatalysis driven by photosynthesis – an integrated bioprocess design for phototrophic biocatalysts

Much success was already achieved for the development of efficient oxyfunctionalization bioprocesses by the application of oxygenases in heterotrophic whole-cell host systems. However, several restrictions such as the technically limited O2 supply and carbohydrate-based electron supply still limit their implementation on an industrial scale concerning production rates and costs. The use of phototrophic organisms as whole-cell biocatalysts for oxygenase-based biotransformations provides an alternative and promising technology for the eco-efficient production of oxyfunctionalized value-added chemicals. While numerous cyanobacterial or microalgal bioprocesses were already developed for CO2-derived fermentations, biotransformation processes relying on the generation of activated reduction equivalents as well as O2-derived from photosynthetic water oxidation are rare. In this context, research mainly focuses on the demonstration of engineered catalysts with emphasis on the production of hydrogen. Yet, an integrated bioprocess design for the application of phototrophic organisms in redox biotransformations beyond the proof-of-concept catalyst development is lacking. This thesis aims at the integrated application of biotechnological methods and strategies for the development of eco-efficient photosynthesis-driven oxyfunctionalization processes. The main research question combines the conceptual evaluation of photosynthetic electron and O2 supply with the technical applicability of cyanobacteria as phototrophic host organisms in a hydrocarbon oxyfunctionalization bioprocess. Using a guide of integrated bioprocess design, biocatalyst, reaction, and process engineering tools are applied for the establishment of new, photosynthesis-driven bioprocesses

Factor Inhibiting HIF\u27s (FIH) Structure Controls O2 Activation and Reactivity

Factor Inhibiting HIF (FIH) is a Fe(II)-αKG dependent oxygenase that acts as a cellular oxygen sensor in humans. FIH regulates the transcriptional activity of the hypoxia-inducible factor-1 (HIF-1a or HIF), a transcription factor responsible cellular O2 homeostasis. Hydroxylation of the target residue HIF-Asn803, found in the C-terminal transactivation domain (CTAD), inactivates HIF-dependent gene expression. Central to FIH’s function is the activation of O2 after CTAD binding. The mechanistic and structural features of FIH leading to tight coupling between CTAD binding and subsequent O2-activation and reactivity are key for efficient O2 sensing. Our mechanistic studies of WT FIH have revealed inverse solvent isotope effects (SIE) in the steady-state rate constants at limiting concentrations of CTAD or αKG providing direct evidence for aquo release during steady-state turnover. Moreover, the inverse SIEs arise due to a rate-limiting O2 activation step. Characterization of FIH’s O2 activation mechanism using a suite of kinetic probes indicates FIH limits its overall activity through αKG decarboxylation. A key aspect of FIH’s catalytic cycle is that binding and positioning of CTAD precedes O2-activation, yet the means by which CTAD binding stimulates O2 activation is not well understood. The steady-state characterization of five FIH-Gln239 variants tested the role of hydrogen bonding and sterics near the target residue. Coupling ratios indicated a disconnect between O2 activation and CTAD hydroxylation in these variants, whereas WT was tightly coupled. This characterization showed that the proper positioning of HIF-Asn803 by FIH-Gln239 is necessary to suppress uncoupled turnover and to support substrate hydroxylation. The facial triad carboxylate of FIH (Asp201) makes a hydrogen bond to the axial aquo ligand that must be displaced O2 activation to occur. Kinetic pKas observed for Asp201-Gly indicate the H-bond gives stability to the Fe-H2O. Increased autohydroxylation rates were observed for Asp201-Glu compared to WT. New O2 dependent chromophores are observed for Asp201-Gly and Asp201-Ala suggesting this H-bond is crucial to controlling O2 reactivity. Overall, the data suggests FIH’s structure controls O2 activation. Gln239 ensures proper positioning of HIF-Asn803 and Asp201provides stability to the Fe-H2O suppressing autohydroxylation. These structural features allow for FIH to efficiently sense O2 to properly regulate HIF mediated gene transcription

Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-β-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.Deutsche Forschungsgemeinschaft AL892/1-4Academy of Finland 125312