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Scientific Reports Jul 2020Lanthanide elements have been recently recognized as "new life metals" yet much remains unknown regarding lanthanide acquisition and homeostasis. In Methylorubrum...
Lanthanide elements have been recently recognized as "new life metals" yet much remains unknown regarding lanthanide acquisition and homeostasis. In Methylorubrum extorquens AM1, the periplasmic lanthanide-dependent methanol dehydrogenase XoxF1 produces formaldehyde, which is lethal if allowed to accumulate. This property enabled a transposon mutagenesis study and growth studies to confirm novel gene products required for XoxF1 function. The identified genes encode an MxaD homolog, an ABC-type transporter, an aminopeptidase, a putative homospermidine synthase, and two genes of unknown function annotated as orf6 and orf7. Lanthanide transport and trafficking genes were also identified. Growth and lanthanide uptake were measured using strains lacking individual lanthanide transport cluster genes, and transmission electron microscopy was used to visualize lanthanide localization. We corroborated previous reports that a TonB-ABC transport system is required for lanthanide incorporation to the cytoplasm. However, cells were able to acclimate over time and bypass the requirement for the TonB outer membrane transporter to allow expression of xoxF1 and growth. Transcriptional reporter fusions show that excess lanthanides repress the gene encoding the TonB-receptor. Using growth studies along with energy dispersive X-ray spectroscopy and transmission electron microscopy, we demonstrate that lanthanides are stored as cytoplasmic inclusions that resemble polyphosphate granules.
Topics: ATP-Binding Cassette Transporters; Alkyl and Aryl Transferases; Aminopeptidases; Bacterial Adhesion; Bacterial Proteins; Cytoplasm; Homeostasis; Lanthanoid Series Elements; Methanol; Methylobacterium extorquens; Microscopy, Electron, Transmission; Mutagenesis
PubMed: 32728125
DOI: 10.1038/s41598-020-69401-4 -
ChemSusChem Oct 2020Power-to-X technologies have the potential to pave the way towards a future resource-secure bioeconomy as they enable the exploitation of renewable resources and CO ....
Power-to-X technologies have the potential to pave the way towards a future resource-secure bioeconomy as they enable the exploitation of renewable resources and CO . Herein, the coupled electrocatalytic and microbial catalysis of the C -polymer precursors mesaconate and 2S-methylsuccinate from CO and electric energy by in situ coupling electrochemical and microbial catalysis at 1 L-scale was developed. In the first phase, 6.1±2.5 mm formate was produced by electrochemical CO reduction. In the second phase, formate served as the substrate for microbial catalysis by an engineered strain of Methylobacterium extorquens AM-1 producing 7±2 μm and 10±5 μm of mesaconate and 2S-methylsuccinate, respectively. The proof of concept showed an overall conversion efficiency of 0.2 % being 0.4 % of the theoretical maximum.
Topics: Carbon Dioxide; Catalysis; Cell Culture Techniques; Electrochemical Techniques; Formates; Fumarates; Maleates; Methylobacterium extorquens; Polymers; Succinates
PubMed: 32658366
DOI: 10.1002/cssc.202001272 -
Journal of the American Chemical Society Jul 2020Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical...
Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.
Topics: Bacterial Proteins; Biocatalysis; Crystallography, X-Ray; Endopeptidases; Iron; Methylobacterium extorquens; Models, Molecular; Molecular Structure; Sulfur
PubMed: 32643933
DOI: 10.1021/jacs.0c02044 -
International Journal of Molecular... Jun 2020YC-XJ1 isolated from desert soil exhibited a diverse degrading ability towards aromatic oxyphenoxypropionic acid esters (AOPPs) herbicide, phthalate esters (PAEs),...
YC-XJ1 isolated from desert soil exhibited a diverse degrading ability towards aromatic oxyphenoxypropionic acid esters (AOPPs) herbicide, phthalate esters (PAEs), organophosphorus flame retardants (OPFRs), chlorpyrifos and phoxim. The genome of YC-XJ1 was sequenced and analyzed systematically. YC-XJ1 contained a large number of exogenous compounds degradation pathways and hydrolase resources. The quizalofop-p-ethyl (QPE) degrading gene and diethyl phthalate (DEP) degrading gene were cloned and expressed. The characteristics of corresponding hydrolases were investigated. The specific activity of recombinant QPEH2 was 0.1 ± 0.02 U mg for QPE with / values of 1.8 ± 0.016 (mM·s). The specific activity of recombinant DEPH1 was 0.1 ± 0.02 U mg for DEP with / values of 0.8 ± 0.02 (mM·s). This work systematically illuminated the metabolic versatility of strain YC-XJ1 via the combination of genomics analysis and laboratory experiments. These results suggested that strain YC-XJ1 with diverse xenobiotics biodegrading capacity was a promising candidate for the bioremediation of polluted sites.
Topics: Amino Acid Sequence; Biodegradation, Environmental; DNA, Bacterial; Genome, Bacterial; Hydrolases; Methylobacteriaceae; Phylogeny; Sequence Homology; Soil Microbiology; Xenobiotics
PubMed: 32580446
DOI: 10.3390/ijms21124436 -
Molecules (Basel, Switzerland) May 2020Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some...
Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from and and a new Fe-type NHase from were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(-toluoyl)propanenitrile. The Co-dependent NHases from and , as well as two Fe-dependent NHases from , were-in addition-able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts.
Topics: Burkholderiaceae; Catalysis; Cobalt; Escherichia coli; Hydro-Lyases; Iron; Metalloproteins; Methylobacteriaceae; Pseudomonas
PubMed: 32481666
DOI: 10.3390/molecules25112521 -
Journal of Agricultural and Food... Jun 2020We previously constructed a heterologous production system for ergothioneine (ERG) in using five ERG biosynthesis genes () from . However, significant amounts of...
We previously constructed a heterologous production system for ergothioneine (ERG) in using five ERG biosynthesis genes () from . However, significant amounts of hercynine (HER), an intermediate of ERG, as ERG were accumulated, suggesting that the reaction of EgtB catalyzing the attachment of γ-glutamylcysteine (γGC) to HER to yield hercynyl-γ-glutamylcysteine sulfoxide was a bottleneck. In this study, we searched for other EgtBs and found many orthologs in diverse microorganisms. Among these, strains possessed EgtBs that catalyze the direct conversion of HER into hercynylcysteine sulfoxide with l-cysteine (l-Cys) as a sulfur donor, in a manner similar to those of acidobacterial CthEgtB and fungal Egt1. An study with recombinant EgtBs from and clearly showed that both enzymes accepted l-Cys but not γGC. We reconstituted the ERG production system in with from ; ERG productivity reached 657 mg L.
Topics: Bacterial Proteins; Betaine; Biosynthetic Pathways; Dipeptides; Ergothioneine; Escherichia coli; Histidine; Metabolic Engineering; Methylobacterium; Sulfoxides
PubMed: 32436380
DOI: 10.1021/acs.jafc.0c01846 -
The Journal of Biological Chemistry Jun 2020The lanthanide elements (Ln), those with atomic numbers 57-63 (excluding promethium, Pm), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF...
The lanthanide elements (Ln), those with atomic numbers 57-63 (excluding promethium, Pm), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs), expanding the range of biological elements and opening novel areas of metabolism and ecology. Other MDHs, known as MxaFIs, are related in sequence and structure to these proteins, yet they instead possess a Ca-PQQ cofactor. An important missing piece of the Ln puzzle is defining what features distinguish enzymes that use Ln-PQQ cofactors from those that do not. Here, using XoxF1 MDH from the model methylotrophic bacterium AM1, we investigated the functional importance of a proposed lanthanide-coordinating aspartate residue. We report two crystal structures of XoxF1, one with and another without PQQ, both with La bound in the active-site region and coordinated by Asp Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp is needed for catalytic function, activity, and La coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La, coordinated Ca but exhibited little or no catalytic activity. We also generated the parallel substitution in ExaF to produce ExaF D319S and found that this variant loses the capacity for efficient ethanol oxidation with La These results provide evidence that a Ln-coordinating aspartate is essential for the enzymatic functions of XoxF MDHs and ExaF EDHs, supporting the notion that sequences of these enzymes, and the genes that encode them, are markers for Ln metabolism.
Topics: Alcohol Oxidoreductases; Amino Acid Substitution; Aspartic Acid; Bacterial Proteins; Biocatalysis; Calcium; Crystallography, X-Ray; Lanthanoid Series Elements; Methanol; Methylobacterium extorquens; Oxidation-Reduction; Structure-Activity Relationship
PubMed: 32366463
DOI: 10.1074/jbc.RA120.013227 -
Biomolecules Apr 2020Motile sp. ME121 and non-motile sp. 32K were isolated from the same soil sample. Interestingly, ME121 was significantly more motile in the coculture of ME121 and 32K...
Motile sp. ME121 and non-motile sp. 32K were isolated from the same soil sample. Interestingly, ME121 was significantly more motile in the coculture of ME121 and 32K than in the monoculture of ME121. This advanced motility of ME121 was also observed in the 32K culture supernatant. A swimming acceleration factor, which we named the K factor, was identified in the 32K culture supernatant, purified, characterized as an extracellular polysaccharide (5-10 kDa), and precipitated with 70% ethanol. These results suggest the possibility that the K factor was directly or indirectly sensed by the flagellar stator, accelerating the flagellar rotation of ME121. To the best of our knowledge, no reports describing an acceleration in motility due to coculture with two or more types of bacteria have been published. We propose a mechanism by which the increase in rotational force of the ME121 flagellar motor is caused by the introduction of the additional stator into the motor by the K factor.
Topics: Bacterial Proteins; Chemical Precipitation; Ethanol; Flagella; Methylobacterium; Monosaccharides; Movement; Rhizobiaceae; Rotation
PubMed: 32316239
DOI: 10.3390/biom10040618 -
Chembiochem : a European Journal of... Sep 2020The family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous...
The family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto-reducing activity, yet negligible imine-reducing capability, and mining the Short-Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active-site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine-reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto-reducing activity was engineered into a promiscuous enzyme with imine-reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the "classical" SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine-reducing enzymes.
Topics: Asparagus Plant; Imines; Ketones; Methylobacterium; Models, Molecular; Molecular Structure; Oxidation-Reduction; Short Chain Dehydrogenase-Reductases
PubMed: 32315494
DOI: 10.1002/cbic.202000233 -
Microbiome Mar 2020Methanol is the second most abundant volatile organic compound in the atmosphere, with the majority produced as a metabolic by-product during plant growth. There is a...
BACKGROUND
Methanol is the second most abundant volatile organic compound in the atmosphere, with the majority produced as a metabolic by-product during plant growth. There is a large disparity between the estimated amount of methanol produced by plants and the amount which escapes to the atmosphere. This may be due to utilisation of methanol by plant-associated methanol-consuming bacteria (methylotrophs). The use of molecular probes has previously been effective in characterising the diversity of methylotrophs within the environment. Here, we developed and applied molecular probes in combination with stable isotope probing to identify the diversity, abundance and activity of methylotrophs in bulk and in plant-associated soils.
RESULTS
Application of probes for methanol dehydrogenase genes (mxaF, xoxF, mdh2) in bulk and plant-associated soils revealed high levels of diversity of methylotrophic bacteria within the bulk soil, including Hyphomicrobium, Methylobacterium and members of the Comamonadaceae. The community of methylotrophic bacteria captured by this sequencing approach changed following plant growth. This shift in methylotrophic diversity was corroborated by identification of the active methylotrophs present in the soils by DNA stable isotope probing using C-labelled methanol. Sequencing of the 16S rRNA genes and construction of metagenomes from the C-labelled DNA revealed members of the Methylophilaceae as highly abundant and active in all soils examined. There was greater diversity of active members of the Methylophilaceae and Comamonadaceae and of the genus Methylobacterium in plant-associated soils compared to the bulk soil. Incubating growing pea plants in a CO atmosphere revealed that several genera of methylotrophs, as well as heterotrophic genera within the Actinomycetales, assimilated plant exudates in the pea rhizosphere.
CONCLUSION
In this study, we show that plant growth has a major impact on both the diversity and the activity of methanol-utilising methylotrophs in the soil environment, and thus, the study contributes significantly to efforts to balance the terrestrial methanol and carbon cycle. Video abstract.
Topics: Alcohol Oxidoreductases; Bacteria; DNA, Bacterial; Genetic Variation; Metagenome; Methanol; Methylobacterium; Phylogeny; Plant Physiological Phenomena; Plants; RNA, Ribosomal, 16S; Rhizosphere; Soil Microbiology
PubMed: 32156318
DOI: 10.1186/s40168-020-00801-4