2-Oxoglutarate derivatives can selectively enhance or inhibit the activity of human oxygenases

Abstract2-Oxoglutarate (2OG) oxygenases are validated agrochemical and human drug targets. The potential for modulating their activity with 2OG derivatives has not been explored, possibly due to concerns regarding selectivity. We report proof-of-principle studies demonstrating selective enhancement or inhibition of 2OG oxygenase activity by 2-oxo acids. The human 2OG oxygenases studied, factor inhibiting hypoxia-inducible transcription factor HIF-α (FIH) and aspartate/asparagine-β-hydroxylase (AspH), catalyze C3 hydroxylations of Asp/Asn-residues. Of 35 tested 2OG derivatives, 10 enhance and 17 inhibit FIH activity. Comparison with results for AspH reveals that 2OG derivatives selectively enhance or inhibit FIH or AspH. Comparison of FIH structures complexed with 2OG derivatives to those for AspH provides insight into the basis of the observed selectivity. 2-Oxo acid derivatives have potential as drugs, for use in biomimetic catalysis, and in functional studies. The results suggest that the in vivo activity of 2OG oxygenases may be regulated by natural 2-oxo acids other than 2OG.

Knock-In Mice Expressing a 15-Lipoxygenating Alox5 Mutant Respond Differently to Experimental Inflammation Than Reported Alox5−/− Mice

Arachidonic acid 5-lipoxygenase (ALOX5) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes. We recently created knock-in mice (Alox5-KI) which express an arachidonic acid 15-lipoxygenating Alox5 mutant instead of the 5-lipoxygenating wildtype enzyme. These mice were leukotriene deficient but exhibited an elevated linoleic acid oxygenase activity. Here we characterized the polyenoic fatty acid metabolism of these mice in more detail and tested the animals in three different experimental inflammation models. In experimental autoimmune encephalomyelitis (EAE), Alox5-KI mice displayed an earlier disease onset and a significantly higher cumulative incidence rate than wildtype controls but the clinical score kinetics were not significantly different. In dextran sodium sulfate-induced colitis (DSS) and in the chronic constriction nerve injury model (CCI), Alox5-KI mice performed like wildtype controls with similar genetic background. These results were somewhat surprising since in previous loss-of-function studies targeting leukotriene biosynthesis (Alox5−/− mice, inhibitor studies), more severe inflammatory symptoms were observed in the EAE model but the degree of inflammation in DSS colitis was attenuated. Taken together, our data indicate that these mutant Alox5-KI mice respond differently in two models of experimental inflammation than Alox5−/− animals tested previously in similar experimental setups.

Exploring the oxygenase function of Form II Rubisco for production of glycolate from CO2

The oxygenase activity of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) converts ribulose-1,5-bisphosphate (RuBP) into 2-phosphoglycolate, which in turn channels into photorespiration, resulting in carbon and energy loss in higher plants. We observed that glycolate can be accumulated extracellularly when two genes encoding the glycolate dehydrogenase of cyanobacteria Synechocystis sp. PCC 6803 were inactivated. This inspired us to explore the oxygenase function of Rubisco for production of glycolate, an important industrial chemical, from CO2 by engineered cyanobacteria. Since the oxygenase activity of Rubisco is generally low in CO2-rich carboxysome of cyanobacteria, we introduced Form II Rubisco, which cannot be assembled in carboxysome, into the cytoplasm of cyanobacteria. Heterologous expression of a Form II Rubisco from endosymbiont of tubeworm Riftia pachyptila (RPE Rubisco) significantly increased glycolate production. We show that the RPE Rubisco is expressed in the cytoplasm. Glycolate production increased upon addition of NaHCO3 but decreased upon supplying CO2. The titer of glycolate reached 2.8 g/L in 18 days, a 14-fold increase compared with the initial strain with glycolate dehydrogenase inactivated. This is also the highest glycolate titer biotechnologically produced from CO2 ever reported. Photosynthetic production of glycolate demonstrated the oxygenase activity of Form II Rubisco can be explored for production of chemicals from CO2.

Oxidation of a Mixture of Polyaromatic Hydrocarbons by a Mixed Culture of Hydrocarbon-Oxidizing Microorganisms

The possibility of biochemical oxidation of polyaromatic hydrocarbon mixtures (PAHs) by the mixed culture of hydrocarbon-oxidizing microorganisms (HOM) in a liquid medium and soil was investigated. The mixed HOM culture was represented by Pseudomonas stutzeri, Pseudomonas putida, Bacillus cereus, and Arthrobacter globiformis genera. It was shown that during HOM cultivation of the microorganisms under study in the liquid medium their number increases from 0.25·104 to 11·108 CFU/ml, which is accompanied by an increase in their oxygenase activity. All PAHs identified were subjected to oxidation from 11.3 to 100%. The results of experiments on biodegradation of PAHs under natural conditions have shown that for 60 days the total utilization of oil products in soils was on the average 65% of the initial contamination. This suggests the prospects for the use of the mixed HOM culture under study for effective biodegradation of PAHs polluting soil and waste waters.

Exploring the Oxygenase Function of Form II Rubisco for Production of Glycolate from CO2

The oxygenase activity of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) converts ribulose-1,5-bisphosphate (RuBP) into 2-phosphoglycolate, which in turn channels into photorespiration, resulting in carbon and energy loss in higher plants. We observed that glycolate can be accumulated extracellularly when two genes encoding the glycolate dehydrogenase of cyanobacteria Synechocystis sp. PCC 6803 were inactivated. This inspired us to explore the oxygenase function of Rubisco for production of glycolate, an important industrial chemical, from CO2 by engineered cyanobacteria. Since the oxygenase activity of Rubisco is generally low in CO2-rich carboxysome of cyanobacteria, we introduced Form II Rubisco, which cannot be assembled in carboxysome, into the cytoplasm of cyanobacteria. Heterologous expression of a Form II Rubisco from endosymbiont of tubeworm Riftia pachyptila (RPE Rubisco) significantly increased glycolate production. We show that the RPE Rubisco is expressed in the cytoplasm. Glycolate production increased upon addition of NaHCO3 but decreased upon supplying CO2. The titer of glycolate reached 2.8 g/L in 18 days, a 14-fold increase compared with the initial strain with glycolate dehydrogenase inactivated. This is also the highest glycolate titer biotechnologically produced from CO2 ever reported. Photosynthetic production of glycolate demonstrated the oxygenase activity of Form II Rubisco can be explored for production of chemicals from CO2.

Downstream Signalling from Molecular Hydrogen

Molecular hydrogen (H2) is now considered part of the suite of small molecules that can control cellular activity. As such, H2 has been suggested to be used in the therapy of diseases in humans and in plant science to enhance the growth and productivity of plants. Treatments of plants may involve the creation of hydrogen-rich water (HRW), which can then be applied to the foliage or roots systems of the plants. However, the molecular action of H2 remains elusive. It has been suggested that the presence of H2 may act as an antioxidant or on the antioxidant capacity of cells, perhaps through the scavenging of hydroxyl radicals. H2 may act through influencing heme oxygenase activity or through the interaction with reactive nitrogen species. However, controversy exists around all the mechanisms suggested. Here, the downstream mechanisms in which H2 may be involved are critically reviewed, with a particular emphasis on the H2 mitigation of stress responses. Hopefully, this review will provide insight that may inform future research in this area.

The impact of photorespiration on plant primary metabolism through metabolic and redox regulation

Photorespiration is an inevitable trait of all oxygenic phototrophs, being the only known metabolic route that converts the inhibitory side-product of Rubisco's oxygenase activity 2-phosphoglycolate (2PG) back into the Calvin–Benson (CB) cycle's intermediate 3-phosphoglycerate (3PGA). Through this function of metabolite repair, photorespiration is able to protect photosynthetic carbon assimilation from the metabolite intoxication that would occur in the present-day oxygen-rich atmosphere. In recent years, much plant research has provided compelling evidence that photorespiration safeguards photosynthesis and engages in cross-talk with a number of subcellular processes. Moreover, the potential of manipulating photorespiration to increase the photosynthetic yield potential has been demonstrated in several plant species. Considering this multifaceted role, it is tempting to presume photorespiration itself is subject to a suite of regulation mechanisms to eventually exert a regulatory impact on other processes, and vice versa. The identification of potential pathway interactions and underlying regulatory aspects has been facilitated via analysis of the photorespiratory mutant phenotype, accompanied by the emergence of advanced omics’ techniques and biochemical approaches. In this mini-review, I focus on the identification of enzymatic steps which control the photorespiratory flux, as well as levels of transcriptional, posttranslational, and metabolic regulation. Most importantly, glycine decarboxylase (GDC) and 2PG are identified as being key photorespiratory determinants capable of controlling photorespiratory flux and communicating with other branches of plant primary metabolism.