Hypoxia-inducible factor prolyl hydroxylase domain 2 (PHD2) is an important oxygen sensor in animals. By using the CO-releasing molecule-2 (CORM-2) as an in situ CO donor, we demonstrate that CO is an inhibitor of PHD2. This report provides further evidence about the emerging role of CO in oxygen sensing and homeostasis.
The present study reported for the first time covalent immobilization of protocatechuate 3,4-dioxygenase (3,4-POD) onto functionalized multi-walled carbon nanotubes (F-MWCNT) for degrading the toxic 3,4-dihydroxybenzoic acid (3,4-DHBA) pollutant in water. The F-MWCNTs had a maximum 3,4-POD loading of 1060 μg/mg. Immobilized 3,4 POD had 44% of relative structural changes to its free configurations. Nevertheless, >90% of relative activity and about 50% of catalytic efficiency were retained to the free enzyme. Immobilized 3,4-POD demonstrated higher alkaline stability and thermostability than the free 3,4-POD. The free and immobilized 3,4-POD lost 82% and 66% of relative activities, respectively after 180 min of incubations at 90 °C. Excellent shelf-life was observed for the immobilized 3,4-POD with residual activity of 56% compared with 41% and 39% of the free 3,4-POD at 4 °C and 25 °C over 30 days storage. Immobilized 3,4-POD showed >60% of catalytic activity retention even after ten-cycle uses, defraying the expenses of free 3,4-POD productions for long term uses. Finally, the immobilized 3,4-POD removed 71% of 3,4-DHBA from water in <4 h, paving its future application for water purification with reduced costs and time.
Molecular oxygen is essential for all multicellular life forms. In humans, the hypoxia-inducible factor (HIF) prolyl hydroxylase domain-containing enzymes (PHDs) serve as important oxygen sensors by regulating the activity of HIF, the master regulator that mediates cellular oxygen homeostasis, in an oxygen-dependent manner. In normoxia, PHDs catalyze the prolyl hydroxylation of HIF, which leads to its degradation and prevents cellular hypoxic response to be triggered. PHDs are current inhibition targets for the potential treatments of a number of diseases. In this chapter, we discuss in vitro and cell-based methods to study the modulation of PHD2, the most important human PHD isoform in normoxia and mild hypoxia. These include the production and purification of recombinant PHD2, the use of mass spectrometry to follow PHD2-catalyzed reactions and the studies of HIF stabilization in cells by immunoblotting.
The gene coding for the oxygenase component, sadA, of 4-aminobenzenesulfonate (4-ABS) 3,4-dioxygenase in Hydrogenophaga sp. PBC was previously identified via transposon mutagenesis. Expression of wild-type sadA in trans restored the ability of the sadA mutant to grow on 4-ABS. The inclusion of sadB and sadD, coding for a putative glutamine-synthetase-like protein and a plant-type ferredoxin, respectively, further improved the efficiency of 4-ABS degradation. Transcription analysis using the gfp promoter probe plasmid showed that sadABD was expressed during growth on 4-ABS and 4-sulfocatechol. Heterologous expression of sadABD in Escherichia coli led to the biotransformation of 4-ABS to a metabolite which shared a similar retention time and UV/vis profile with 4-sulfocatechol. The putative reductase gene sadC was isolated via degenerate PCR and expression of sadC and sadABD in E. coli led to maximal 4-ABS biotransformation. In E. coli, the deletion of sadB completely eliminated dioxygenase activity while the deletion of sadC or sadD led to a decrease in dioxygenase activity. Phylogenetic analysis of SadB showed that it is closely related to the glutamine-synthetase-like proteins involved in the aniline degradation pathway. This is the first discovery, to our knowledge, of the functional genetic components for 4-ABS aromatic ring hydroxylation in the bacterial domain.
Pseudomonas alcaligenes NCIMB 9867 (strain P25X) is known to synthesize two isofunctional gentisate 1,2-dioxygenases (GDO; EC 1.13.11.4) as well as other enzymes involved in the degradation of xylenols and cresols via the gentisate pathway. The hbzE gene encoding what is possibly the strictly inducible gentisate 1,2-dioxygenase II (GDO-II) was cloned, overexpressed and purified as a hexahistidine fusion protein from Escherichia coli. Active recombinant GDO-II had an estimated molecular mass of 150kDa and is likely a tetrameric protein with a subunit mass of approximately 40kDa, similar to the previously characterized gentisate 1,2-dioxygenase I (GDO-I) encoded by xlnE. However, GDO-II was unable to utilize gentisate that is substituted at the carbon-4 position, unlike GDO-I which had broader substrate specificity. GDO-II also possessed different kinetic characteristics when compared to GDO-I. The hbzE-encoded GDO-II shared higher sequence identities (53%) with GDOs from Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2, compared with only 35% identity with the xlnE-encoded GDO-I. The hbzE gene was found to be part of a cluster of nine genes including the putative regulatory gene designated hbzR, which encodes an LysR-type regulator and is divergently transcribed from the other genes of the hbzHIJKLFED cluster.
Bioremediation offers a sustainable approach for removal of polycyclic aromatic hydrocarbons (PAHs) from the environment; however, information regarding the microbial communities involved remains limited. In this study, microbial community dynamics and the abundance of the key gene (PAH-RHDα) encoding a ring hydroxylating dioxygenase involved in PAH degradation were examined during degradation of phenanthrene in a podzolic soil from the site of a former timber treatment facility. The 10,000-fold greater abundance of this gene associated with Gram-positive bacteria found in phenanthrene-amended soil compared to unamended soil indicated the likely role of Gram-positive bacteria in PAH degradation. In contrast, the abundance of the Gram-negative PAHs-RHDα gene was very low throughout the experiment. While phenanthrene induced increases in the abundance of a small number of OTUs from the Actinomycetales and Sphingomonadale, most of the remainder of the community remained stable. A single unclassified OTU from the Micrococcaceae family increased ~20-fold in relative abundance, reaching 32% of the total sequences in amended microcosms on day 7 of the experiment. The relative abundance of this same OTU increased 4.5-fold in unamended soils, and a similar pattern was observed for the second most abundant PAH-responsive OTU, classified into the Sphingomonas genus. Furthermore, the relative abundance of both of these OTUs decreased substantially between days 7 and 17 in the phenanthrene-amended and control microcosms. This suggests that their opportunistic phenotype, in addition to likely PAH-degrading ability, was determinant in the vigorous growth of dominant PAH-responsive OTUs following phenanthrene amendment. This study provides new information on the temporal response of soil microbial communities to the presence and degradation of a significant environmental pollutant, and as such has the potential to inform the design of PAH bioremediation protocols.
A white-rot fungus of Polyporus sp. S133 was isolated from an oil-polluted soil. The metabolism of pyrene by this fungus was investigated in liquid medium with 5 mg of the compound. Depletion of pyrene was evident during the 30-day growth period and was 21% and 90%, respectively, in cometabolism and metabolism of pyrene alone. Pyrene was absorbed to fungal cells or biodegraded to form simpler structural compounds. Seventy-one percent of eliminated pyrene was transformed by Polyporus sp. S133 into other compounds, whereas only 18% was absorbed in the fungal cell. The effects of pH and temperature on biomass production of Polyporus sp. S133 for pyrene were examined; the properties of laccase and 1,2-dioxygenase produced by Polyporus sp. S133 during pyrene degradation were investigated. The optimal values of pH were 3, 5, and 4 for laccase, 1,2-dioxygenase, and biomass production, respectively, whereas the optimal values of temperature were 25 °C for laccase and 50 °C for 1,2-dioxygenase and biomass production. Under optimal conditions, pyrene was mainly metabolized to 1-hydroxypyrene and gentisic acid. The structure of 1-hydroxypyrene and gentisic acid was determined by gas chromatography-mass spectrometry after identification using thin-layer chromatography.
Phenanthrene degradation by Polyporus sp. S133, a new phenanthrene-degrading strain, was investigated in this work. The analysis of degradation was performed by calculation of the remaining phenanthrene by gas chromatography-mass spectrometry. When cells were grown in phenanthrene culture after 92 h, all but 200 and 250 mg/l of the phenanthrene had been degraded. New metabolic pathways of phenanthrene and a better understanding of the phenoloxidases and dioxygenase mechanism involved in degradation of phenanthrene were explored in this research. The mechanism of degradation was determined through identification of the several metabolites; 9,10-phenanthrenequinone, 2,2'-diphenic acid, salicylic acid, and catechol. 9,10-Oxidation and ring cleavage to give 9,10-phenanthrenequinone is the major fate of phenanthrene in ligninolytic Polyporus sp. S133. The identification of 2,2'-diphenic acid in culture extracts indicates that phenanthrene was initially attacked through dioxigenation at C9 and C10 to give cis-9,10-dihydrodiol. Dehydrogenation of phenanthrene-cis-9,10-dihydrodiol to produce the corresponding diol, followed by ortho-cleavage of the oxygenated ring, produced 2,2'-diphenic acid. Several enzymes (manganese peroxidase, lignin peroxidase, laccase, 1,2-dioxygenase, and 2,3-dioxygenase) produced by Polyporus sp. S133 was detected during the incubation. The highest level of activity was shown at 92 h of culture.
The coexisting mechanism of a synthetic bacterial community (SBC) was investigated to better understand how to manage microbial communities. The SBC was constructed with three kinds of phenol-utilizing bacteria, Pseudomonas sp. LAB-08, Comamonas testosteroni R2, and Cupriavidus sp. P-10, under chemostat conditions supplied with phenol as a sole carbon and energy source. Population densities of all strains were monitored by real-time quantitative PCR (qPCR) targeting the gene encoding the large subunit of phenol hydroxylase. Although the supply of phenol was stopped to allow perturbation in the SBC, all of the strains coexisted and the degradation of phenol was maintained for more than 800 days. The qPCR analyses showed that strains LAB-08 and R2 became dominant simultaneously, whereas strain P-10 was a minor population. This phenomenon was observed before and after the phenol-supply stoppage. The kinetic parameters for phenol of the SBC changed before and after the phenol-supply stoppage, which suggests a change in functional roles of strains in the SBC. Transcriptional levels of phenol hydroxylase and catechol dioxygenases of three strains were monitored by reverse-transcription qPCR (RT-qPCR). The RT-qPCR analyses revealed that all strains shared phenol and survived independently before the phenol-supply stoppage. After the stoppage, strain P-10 would incur the cost for degradation of phenol and catechol, whereas strains LAB-08 and R2 seemed to be cheaters using metabolites, indicating the development of the metabolic network. These results indicated that it is important for the management and redesign of microbial communities to understand the metabolism of bacterial communities.
Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.
Hypoxia inducible factor (HIF)-prolyl hydroxylase (PHD) inhibitors are shown to be protective in several models of inflammatory bowel disease (IBD). However, these non-selective inhibitors are known to inhibit all the three isoforms of PHD, i.e. PHD-1, PHD-2 and PHD-3. In the present report, we investigated the associated changes in levels of PHDs during the development and recovery of chemically induced colitis in mice. The results indicated that in the experimental model of murine colitis, levels of both, PHD-1 and PHD-2 were found to be increased with the progression of the disease; however, the level of PHD-3 remained the same in group of healthy controls and mice with colitis. Thus, the findings advocated that inhibitors, which inhibited all three isoforms of PHD could not be ideal therapeutics for IBD since PHD-3 is required for normal gut function. Hence, this necessitates the development of new compounds capable of selectively inhibiting PHD-1 and PHD-2 for effective treatment of IBD.
Bacteria belonging to the genus Novosphingobium are known to be metabolically versatile and occupy different ecological niches. In the absence of genomic data and/or analysis, knowledge of the bacteria that belong to this genus is currently limited to biochemical characteristics. In this study, we analyzed the whole genome sequencing data of six bacteria in the Novosphingobium genus and provide evidence to show the presence of genes that are associated with salt tolerance, cell-cell signaling and aromatic compound biodegradation phenotypes. Additionally, we show the taxonomic relationship between the sequenced bacteria based on phylogenomic analysis, average amino acid identity (AAI) and genomic signatures.
Molecular dissection of inborn errors of immunity can help to elucidate the nonredundant functions of individual genes. We studied 3 children with an immune dysregulation syndrome of susceptibility to infection, lymphadenopathy, hepatosplenomegaly, developmental delay, autoimmunity, and lymphoma of B-cell (n = 2) or T-cell (n = 1) origin. All 3 showed early autologous T-cell reconstitution following allogeneic hematopoietic stem cell transplantation. By whole-exome sequencing, we identified rare homozygous germline missense or nonsense variants in a known epigenetic regulator of gene expression: ten-eleven translocation methylcytosine dioxygenase 2 (TET2). Mutated TET2 protein was absent or enzymatically defective for 5-hydroxymethylating activity, resulting in whole-blood DNA hypermethylation. Circulating T cells showed an abnormal immunophenotype including expanded double-negative, but depleted follicular helper, T-cell compartments and impaired Fas-dependent apoptosis in 2 of 3 patients. Moreover, TET2-deficient B cells showed defective class-switch recombination. The hematopoietic potential of patient-derived induced pluripotent stem cells was skewed toward the myeloid lineage. These are the first reported cases of autosomal-recessive germline TET2 deficiency in humans, causing clinically significant immunodeficiency and an autoimmune lymphoproliferative syndrome with marked predisposition to lymphoma. This disease phenotype demonstrates the broad role of TET2 within the human immune system.