Microbial Degradation of Pyridine: a Complete Pathway in Arthrobacter sp. Strain 68b Deciphered.

Pyridine and its derivatives constitute the majority of heterocyclic aromatic compounds that occur largely as a result of human activities and contribute to environmental pollution. It is known that they can be degraded by various bacteria in the environment; however, the degradation of unsubstituted pyridine has not yet been completely resolved. In this study, we present data on the pyridine catabolic pathway in Arthrobacter sp. strain 68b at the level of genes, enzymes, and metabolites. The pyr gene cluster, responsible for the degradation of pyridine, was identified in a catabolic plasmid, p2MP. The pathway of pyridine metabolism consisted of four enzymatic steps and ended by the formation of succinic acid. The first step in the degradation of pyridine proceeds through a direct ring cleavage catalyzed by a two-component flavin-dependent monooxygenase system, encoded by pyrA (pyridine monooxygenase) and pyrE genes. The genes pyrB, pyrC, and pyrD were found to encode (Z)-N-(4-oxobut-1-enyl)formamide dehydrogenase, amidohydrolase, and succinate semialdehyde dehydrogenase, respectively. These enzymes participate in the subsequent steps of pyridine degradation. The metabolites of these enzymatic reactions were identified, and this allowed us to reconstruct the entire pyridine catabolism pathway in Arthrobacter sp. 68b.IMPORTANCE The biodegradation pathway of pyridine, a notorious toxicant, is relatively unexplored, as no genetic data related to this process have ever been presented. In this paper, we describe the plasmid-borne pyr gene cluster, which includes the complete set of genes responsible for the degradation of pyridine. A key enzyme, the monooxygenase PyrA, which is responsible for the first step of the catabolic pathway, performs an oxidative cleavage of the pyridine ring without typical activation steps such as reduction or hydroxylation of the heterocycle. This work provides new insights into the metabolism of N-heterocyclic compounds in nature.

Nanotubes from bacteriophage tail sheath proteins: internalisation by cancer cells and macrophages.

Bionanoparticles comprised of naturally occurring monomers are gaining interest in the development of novel drug transportation systems. Here we report on the stabilisation, cellular uptake, and macrophage clearance of nanotubes formed from the self-assembling gp053 tail sheath protein of the vB_EcoM_FV3 bacteriophage. To evaluate the potential of the bacteriophage protein-based nanotubes as therapeutic nanocarriers, we investigated their internalisation into colorectal cancer cell lines and professional macrophages that may hinder therapeutic applications by clearing nanotube carriers. We fused the bacteriophage protein with a SNAP-tag self-labelling enzyme and demonstrated that its activity is retained in assembled nanotubes, indicating that such carriers can be applied to deliver therapeutic biomolecules. Under physiological conditions, the stabilisation of the nanotubes by PEGylation was required to prevent aggregation and yield a stable solution with uniform nano-sized structures. Colorectal carcinoma cells from primary and metastatic tumours internalized SNAP-tag-carrying nanotubes with different efficiencies. The nanotubes entered HCT116 cells via dynamin-dependent and SW480 cells - via dynamin- and clathrin-dependent pathways and were accumulated in lysosomes. Meanwhile, peritoneal macrophages phagocytosed the nanotubes in a highly efficient manner through actin-dependent mechanisms. Macrophage clearance of nanotubes was enhanced by inflammatory activation but was dampened in macrophages isolated from aged animals. Altogether, our results demonstrate that gp053 nanotubes retained the cargo's enzymatic activity post-assembly and had the capacity to enter cancer cells. Furthermore, we emphasise the importance of evaluating the nanocarrier clearance by immune cells under conditions mimicking a cancerous environment.

Studies of dimethylglycine oxidase isoenzymes in Arthrobacter globiformis cells.

Glycine betaine (GB) could be used by Arthrobacter globiformis cells as a sole carbon source. The cells took up this molecule in the low as well as in the high salinity medium. Addition of GB to the mineral medium with high salt concentration revealed that GB was also used as an osmoprotectant. Dimethylglycine oxidase (DMGO) was involved in the catabolism of GB. Two genes for DMGO were detected in a cloned 26267 bp fragment of A. globiformis DNA. The genes involved in the tetrahydrofolate-dependent assimilation of methyl groups were located nearby the two of DMGO genes. Both cloned A. globiformis DMGO were active. The activity of DMGO was detected in A. globiformis cells and it depended on the addition of GB and the salinity of the medium. Reverse transcription-PCR demonstrated that the addition of GB influenced the transcription of dmg genes.

Characterization of a Yellow Laccase from Botrytis cinerea 241.

Typical laccases have four copper atoms, which form three different copper centers, of which the T1 copper is responsible for the blue color of the enzyme and gives it a characteristic absorbance around 610 nm. Several laccases have unusual spectral properties and are referred to as yellow or white laccases. Only two yellow laccases from the Ascomycota phylum have been described previously, and only one amino acid sequence of those enzymes is available. A yellow laccase Bcl1 from Botrytis cinerea strain 241 has been identified, purified and characterized in this work. The enzyme appears to be a dimer with a molecular mass of 186 kDa. The gene encoding the Bcl1 protein has been cloned, and the sequence analysis shows that the yellow laccase Bcl1 is phylogenetically distinct from other known yellow laccases. In addition, a comparison of amino acid sequences, and 3D modeling shows that the Bcl1 laccase lacks a conservative tyrosine, which is responsible for absorption quenching at 610 nm in another yellow asco-laccase from Sclerotinia sclerotiorum. High thermostability, high salt tolerance, broad substrate specificity, and the ability to decolorize dyes without the mediators suggest that the Bcl1 laccase is a potential enzyme for various industrial applications.

Functionalized Protein Nanotubes Based on the Bacteriophage vB_KleM-RaK2 Tail Sheath Protein.

We report on the construction of functionalized nanotubes based on tail sheath protein 041 from vB_KleM-RaK2 bacteriophage. The truncated 041 protein (041Δ200) was fused with fluorescent proteins GFP and mCherry or amidohydrolase YqfB. The generated chimeric proteins were successfully synthesized in E. coli BL21 (DE3) cells and self-assembled into tubular structures. We detected the fluorescence of the structures, which was confirmed by stimulated emission depletion microscopy. When 041Δ200GFP and 041Δ200mCherry were coexpressed in E. coli BL21 (DE3) cells, the formed nanotubes generated Förster resonance energy transfer, indicating that both fluorescent proteins assemble into a single nanotube. Chimeric 041Δ200YqfB nanotubes possessed an enzymatic activity, which was confirmed by hydrolysis of N4-acetyl-2'-deoxycytidine. The enzymatic properties of 041Δ200YqfB were similar to those of a free wild-type YqfB. Hence, we conclude that 041-based chimeric nanotubes have the potential for the development of delivery vehicles and targeted imaging and are applicable as scaffolds for biocatalysts.

Engineering of a chromogenic enzyme screening system based on an auxiliary indole-3-carboxylic acid monooxygenase.

Here, we present a proof-of-principle for a new high-throughput functional screening of metagenomic libraries for the selection of enzymes with different activities, predetermined by the substrate being used. By this approach, a total of 21 enzyme-coding genes were selected, including members of xanthine dehydrogenase, aldehyde dehydrogenase (ALDH), and amidohydrolase families. The screening system is based on a pro-chromogenic substrate, which is transformed by the target enzyme to indole-3-carboxylic acid. The later compound is converted to indoxyl by a newly identified indole-3-carboxylate monooxygenase (Icm). Due to the spontaneous oxidation of indoxyl to indigo, the target enzyme-producing colonies turn blue. Two types of pro-chromogenic substrates have been tested. Indole-3-carboxaldehydes and the amides of indole-3-carboxylic acid have been applied as substrates for screening of the ALDHs and amidohydrolases, respectively. Both plate assays described here are rapid, convenient, easy to perform, and adaptable for the screening of a large number of samples both in Escherichia coli and Rhodococcus sp. In addition, the fine-tuning of the pro-chromogenic substrate allows screening enzymes with the desired substrate specificity.

Functionalization of α-synuclein fibrils.

The propensity of peptides and proteins to form self-assembled structures has very promising applications in the development of novel nanomaterials. Under certain conditions, amyloid protein α-synuclein forms well-ordered structures - fibrils, which have proven to be valuable building blocks for bionanotechnological approaches. Herein we demonstrate the functionalization of fibrils formed by a mutant α-synuclein that contains an additional cysteine residue. The fibrils have been biotinylated via thiol groups and subsequently joined with neutravidin-conjugated gold nanoparticles. Atomic force microscopy and transmission electron microscopy confirmed the expected structure - nanoladders. The ability of fibrils (and of the additional components) to assemble into such complex structures offers new opportunities for fabricating novel hybrid materials or devices.

Construction of Escherichia coli-Arthrobacter-Rhodococcus shuttle vectors based on a cryptic plasmid from Arthrobacter rhombi and investigation of their application for functional screening.

A cryptic plasmid from Arthrobacter rhombi PRH1, designated as pPRH, was sequenced and characterized. It was 5000 bp in length with a G+C content of 66 mol%. The plasmid pPRH was predicted to encode six putative open reading frames (ORFs), in which ORF2 and ORF3 formed the minimal replicon of plasmid pPRH and shared 55-61% and 60-69% homology, respectively, with the RepA and RepB proteins of reported rhodococcal plasmids. Sequence analysis revealed a typical ColE2-type ori located 45 bp upstream of the gene repA. Sequence and phylogenetic analysis led to the conclusion that pPRH is a representative of a novel group of pAL5000 subfamily of ColE2 family plasmids. Three shuttle vectors pRMU824, pRMU824Km and pRMU824Tc, encoding chloramphenicol resistance, were constructed. The latter two harboured additional antibiotic resistance genes kan and tet, respectively. All vectors successfully replicated in Escherichia coli, Arthrobacter and Rhodococcus spp. The vector pRMU824Km was employed for functional screening of 2-hydroxypyridine catabolism encoding genes from Arthrobacter sp. PY22. Sequence analysis of the cloned 6-kb DNA fragment revealed eight putative ORFs, among which hpyB gene encoded a putative monooxygenase.

Probing reactivity of PQQ-dependent carbohydrate dehydrogenases using artificial electron acceptor.

The kinetic parameters of carbohydrate oxidation catalyzed by Acinetobacter calcoaceticus pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) and Escherichia coli PQQ-dependent aldose sugar dehydrogenase (ASDH) were determined using various electron acceptors. The radical cations of organic compounds and 2,6-dichlorophenolindophenol are the most reactive with both enzymes in presence of glucose. The reactivity of dioxygen with ASDH is low; the bimolecular constant k (ox) = 660 M(-1) s(-1), while GDH reactivity with dioxygen is even less. The radical cation of 3-(10H-phenoxazin-10-yl)propionic acid was used as electron acceptor for reduced enzyme in the study of dehydrogenases carbohydrates specificity. Mono- and disaccharide reactivity with GDH is higher than the reactivity of oligosaccharides. For ASDH, the reactivity increased with the carbohydrate monomer number increase. The specificity of quinoproteins was compared with specificity of flavoprotein Microdochium nivale carbohydrate oxidase due to potential enzymes application for lactose oxidation.

α-synuclein reactive antibodies as diagnostic biomarkers in blood sera of Parkinson's disease patients.

BACKGROUND: Auto-antibodies with specificity to self-antigens have been implicated in a wide variety of neurological diseases, including Parkinson's (PD) and Alzheimer's diseases, being sensitive indicators of neurodegeneration and focus for disease prevention. Of particular interest are the studies focused on the auto-immune responses to amyloidogenic proteins associated with diseases and their applications in therapeutic treatments such as vaccination with amyloid antigens and antibodies in PD, Alzheimer's disease and potentially other neurodegeneration ailments. METHODOLOGY/PRINCIPAL FINDINGS: Generated auto-antibodies towards the major amyloidogenic protein involved in PD Lewy bodies--α-synuclein and its amyloid oligomers and fibrils were measured in the blood sera of early and late PD patients and controls by using ELISA, Western blot and Biacore surface plasmon resonance. We found significantly higher antibody levels towards monomeric α-synuclein in the blood sera of PD patients compared to controls, though the responses decreased with PD progression (P<0.0001). This indicates potential protective role of autoimmunity in maintaining the body homeostasis and clearing protein species whose disbalance may lead to amyloid assembly. There were no noticeable immune responses towards amyloid oligomers, but substantially increased levels of IgGs towards α-synuclein amyloid fibrils both in PD patients and controls, which subsided with the disease progression (P<0.0001). Pooled IgGs from PD patients and controls interacted also with the amyloid fibrils of Aß (1-40) and hen lysozyme, however the latter were recognized with lower affinity. This suggests that IgGs bind to the generic amyloid conformational epitope, displaying higher specificity towards human amyloid species associated with neurodegeneration. CONCLUSIONS/SIGNIFICANCE: Our findings may suggest the protective role of autoimmunity in PD and therefore immune reactions towards PD major amyloid protein--α-synuclein can be of value in the development of treatment and diagnostic strategies, especially during the early disease stages.

Expression and purification of active recombinant equine lysozyme in Escherichia coli.

Equine lysozyme (EL) is a calcium (Ca)-binding lysozyme and is an intermediary link between non-Ca-binding C-type lysozyme and alpha-lactalbumin. The feature of lysozymes to assemble into the fibrils has recently gained considerable attention for the investigation of the functional properties of these proteins. To study the structural and functional properties of EL, a synthetic gene was cloned and EL was overexpressed in Escherichia coli as a fused protein. The His-tagged recombinant EL was accumulated as inclusion bodies. Up to 50 mg/l of the recombinant EL could be achieved after purification by Ni(2+) affinity chromatography, refolding in the presence of arginine, CM-Sepharose column purification following TEV protease cleavage. The purified protein was functionally active, as determined by the lysozyme activity, proving the proper folding of protein. The purified lysozyme was used for the oligomerisation studies. The protein formed amyloid fibrils during incubation in acidic pH and elevated temperature. The recombinant EL forms two types of fibrils: ring shaped and linear, similar to the native EL.