Multiple Oxidoreductases Research Articles

Activation of oxidoreductases by the formation of enzyme assembly

Biological properties of protein molecules depend on their interaction with other molecules, and enzymes are no exception. Enzyme activities are controlled by their interaction with other molecules in living cells. Enzyme activation and their catalytic properties in the presence of different types of polymers have been studied in vitro, although these studies are restricted to only a few enzymes. In this study, we show that addition of poly-l-lysine (PLL) can increase the enzymatic activity of multiple oxidoreductases through formation of enzyme assemblies. Oxidoreductases with an overall negative charge, such as l-lactate oxidase, d-lactate dehydrogenase, pyruvate oxidase, and acetaldehyde dehydrogenase, each formed assemblies with the positively charged PLL via electrostatic interactions. The enzyme activities of these oxidoreductases in the enzyme assemblies were several-folds higher than those of the enzyme in their natural dispersed state. In the presence of PLL, the turnover number (kcat) improved for all enzymes, whereas the decrease in Michaelis constant (KM) was enzyme dependent. This type of enzyme function regulation through the formation of assemblies via simple addition of polymers has potential for diverse applications, including various industrial and research purposes.

Self-Sufficient Reusable Biocatalytic System Outfitted with Multiple Oxidoreductases and Flexible Polypeptide-Based Cofactor Swing Arms

Oxidoreductases are widely used in value-added chemical synthesis. Since many oxidoreductases require the consumption of expensive cofactors, multi-enzyme cascade reactions, including cofactor regeneration, have been developed. Although enzymes are frequently immobilized for industrial processes, the immobilization of the small molecule cofactor is challenging. Here, we demonstrate that the co-immobilization of a swing arm cofactor and multiple oxidoreductases can overcome these cofactor limitations. We chose a very flexible protein, elastin-like polypeptide (ELP), fused to a strep-tag II as a genetically encoded swing arm. The Escherichia coli expressed and purified swing arm was conjugated with NAD+ to generate a cofactor swing arm (ELP-NAD+). The cofactor swing arm was co-immobilized on a streptavidin resin with two oxidoreductases fused to a strep-tag II: glucose dehydrogenase (GDH) and mannitol dehydrogenase (MDH). We observed a substantial d-mannitol production (about 1.6 mM) in the three-component (GDH, MDH, and ELP-NAD+) co-immobilized microreactor. The three-component co-immobilized system showed 2 and 3 times higher d-mannitol production than the one with free three components and another with the immobilized enzymes and free cofactor, respectively. Moreover, the product could be easily separated from the co-immobilized reactor and the activity was retained when applied for repeated batch reactions (> seven cycles). These results demonstrate that the genetically encoded cofactor swing arm can be stably co-immobilized with multiple oxidoreductases on a solid support and it can maintain reactivity as a cofactor because of the flexible ELP swing arm.

Self-Sufficient Reusable Microreactor Outfitted with Multiple Oxidoreductases and Flexible Polypeptide-Based Cofactor Swing Arms

Self-Sufficient Reusable Microreactor Outfitted with Multiple Oxidoreductases and Flexible Polypeptide-Based Cofactor Swing Arms

2H/1H variation in microbial lipids is controlled by NADPH metabolism

The hydrogen-isotopic compositions (2H/1H ratios) of lipids in microbial heterotrophs are known to vary enormously, by at least 40% (400‰) relative. This is particularly surprising, given that most C-bound H in their lipids appear to derive from the growth medium water, rather than from organic substrates, implying that the isotopic fractionation between lipids and water is itself highly variable. Changes in the lipid/water fractionation are also strongly correlated with the type of energy metabolism operating in the host. Because lipids are well preserved in the geologic record, there is thus significant potential for using lipid 2H/1H ratios to decipher the metabolism of uncultured microorganisms in both modern and ancient ecosystems. But despite over a decade of research, the precise mechanisms underlying this isotopic variability remain unclear. Differences in the kinetic isotope effects (KIEs) accompanying NADP+ reduction by dehydrogenases and transhydrogenases have been hypothesized as a plausible mechanism. However, this relationship has been difficult to prove because multiple oxidoreductases affect the NADPH pool simultaneously. Here, we cultured five diverse aerobic heterotrophs, plus five Escherichia coli mutants, and used metabolic flux analysis to show that 2H/1H fractionations are highly correlated with fluxes through NADP+-reducing and NADPH-balancing reactions. Mass-balance calculations indicate that the full range of 2H/1H variability in the investigated organisms can be quantitatively explained by varying fluxes, i.e., with constant KIEs for each involved oxidoreductase across all species. This proves that lipid 2H/1H ratios of heterotrophic microbes are quantitatively related to central metabolism and provides a foundation for interpreting 2H/1H ratios of environmental lipids and sedimentary hydrocarbons.

Preparation and evaluation of a polymer–metal–enzyme hybrid nanowire for the immobilization of multiple oxidoreductases

AbstractBACKGROUNDThe immobilization of multiple oxidoreductases has attracted a lot of research interest for its industrial application in eco‐friendly production. Here, a novel immobilization strategy was developed using manganese ion chelated linear polyethyleneimine as artificial hybrid scaffold that allowed self‐assembly of bifunctional oxidoreductase (glycerol dehydrogenase‐NADH oxidase) via multipoint coordination interaction.RESULTThe immobilization formed a highly ordered polymer–metal–enzyme complex structure shaped like a leaf vein with 200‐nm‐wide nanowires. Preliminary results showed that the immobilization could not only retain but substantially increased the biological activity of the immobilized enzyme by 8.2‐fold. The equilibrium concentration of the reaction product DHA also was increased by 4.5‐fold compared with that of the free enzyme, exhibiting higher elimination efficiency of product inhibition compared with other enzyme immobilization strategy using nanoparticles or activated agarose beads. At the same time, linear polyethyleneimine (LPEI)–manganese scaffolds could prevent enzyme subunit disassociation and stabilize the active multimeric form, which improved catalytic condition adaption and thermal stability of target enzyme.CONCLUSIONThese results indicated a great potential of polymer–metal–enzyme hybrid in the enzyme immobilization and have provided a simple and efficient strategy for the immobilization of multiple oxidoreductases. © 2018 Society of Chemical Industry

NapA Mediates a Redox Regulation of the Antioxidant Response, Carbon Utilization and Development in Aspergillus nidulans.

The redox-regulated transcription factors (TFs) of the bZIP AP1 family, such as yeast Yap1 and fission yeast Pap1, are activated by peroxiredoxin proteins (Prxs) to regulate the antioxidant response. Previously, Aspergillus nidulans mutants lacking the Yap1 ortholog NapA have been characterized as sensitive to H2O2 and menadione. Here we study NapA roles in relation to TFs SrrA and AtfA, also involved in oxidant detoxification, showing that these TFs play different roles in oxidative stress resistance, catalase gene regulation and development, during A. nidulans life cycle. We also uncover novel NapA roles in repression of sexual development, normal conidiation, conidial mRNA accumulation, and carbon utilization. The phenotypic characterization of ΔgpxA, ΔtpxA, and ΔtpxB single, double and triple peroxiredoxin mutants in wild type or ΔnapA backgrounds shows that none of these Prxs is required for NapA function in H2O2 and menadione resistance. However, these Prxs participate in a minor NapA-independent H2O2 resistance pathway and NapA and TpxA appear to regulate conidiation along the same route. Using transcriptomic analysis we show that during conidial development NapA-dependent gene expression pattern is different from canonical oxidative stress patterns. In the course of conidiation, NapA is required for regulation of at least 214 genes, including ethanol utilization genes alcR, alcA and aldA, and large sets of genes encoding proteins involved in transcriptional regulation, drug detoxification, carbohydrate utilization and secondary metabolism, comprising multiple oxidoreductases, membrane transporters and hydrolases. In agreement with this, ΔnapA mutants fail to grow or grow very poorly in ethanol, arabinose or fructose as sole carbon sources. Moreover, we show that NapA nuclear localization is induced not only by oxidative stress but also by growth in ethanol and by carbon starvation. Together with our previous work, these results show that SakA-AtfA, SrrA and NapA oxidative stress-sensing pathways regulate essential aspects of spore physiology (i.e., cell cycle arrest, dormancy, drug production and detoxification, and carbohydrate utilization).

Integration of Artificial Photosynthesis System for Enhanced Electronic Energy-Transfer Efficacy: A Case Study for Solar-Energy Driven Bioconversion of Carbon Dioxide to Methanol.

Biocatalyzed artificial photosynthesis systems provide a promising strategy to store solar energy in a great variety of chemicals. However, the lack of direct interface between the light-capturing components and the oxidoreductase generally hinders the trafficking of the chemicals and photo-excited electrons into the active center of the redox biocatalysts. To address this problem, a completely integrated artificial photosynthesis system for enhanced electronic energy-transfer efficacy is reported by combining co-axial electrospinning/electrospray and layer-by-layer (LbL) self-assembly. The biocatalysis part including multiple oxidoreductases and coenzymes NAD(H) was in situ encapsulated inside the lumen polyelectrolyte-doped hollow nanofibers or microcapsules fabricated via co-axial electrospinning/electrospray; while the precise and spatial arrangement of the photocatalysis part, including electron mediator and photosensitizer for photo-regeneration of the coenzyme, was achieved by ion-exchange interaction-driven LbL self-assembly. The feasibility and advantages of this integrated artificial photosynthesis system is fully demonstrated by the catalyzed cascade reduction of CO2 to methanol by three dehydrogenases (formate, formaldehyde, and alcohol dehydrogenases), incorporating the photo-regeneration of NADH under visible-light irradiation. Compared to solution-based systems, the methanol yield increases from 35.6% to 90.6% using the integrated artificial photosynthesis. This work provides a novel platform for the efficient and sustained production of a broad range of chemicals and fuels from sunlight.

The role of oxidoreductases in determining the function of the neisserial lipid A phosphoethanolamine transferase required for resistance to polymyxin.

The decoration of the lipid A headgroups of the lipooligosaccharide (LOS) by the LOS phosphoethanolamine (PEA) transferase (LptA) in Neisseria spp. is central for resistance to polymyxin. The structure of the globular domain of LptA shows that the protein has five disulphide bonds, indicating that it is a potential substrate of the protein oxidation pathway in the bacterial periplasm. When neisserial LptA was expressed in Escherichia coli in the presence of the oxidoreductase, EcDsbA, polymyxin resistance increased 30-fold. LptA decorated one position of the E. coli lipid A headgroups with PEA. In the absence of the EcDsbA, LptA was degraded in E. coli. Neisseria spp. express three oxidoreductases, DsbA1, DsbA2 and DsbA3, each of which appear to donate disulphide bonds to different targets. Inactivation of each oxidoreductase in N. meningitidis enhanced sensitivity to polymyxin with combinatorial mutants displaying an additive increase in sensitivity to polymyxin, indicating that the oxidoreductases were required for multiple pathways leading to polymyxin resistance. Correlates were sought between polymyxin sensitivity, LptA stability or activity and the presence of each of the neisserial oxidoreductases. Only meningococcal mutants lacking DsbA3 had a measurable decrease in the amount of PEA decoration on lipid A headgroups implying that LptA stability was supported by the presence of DsbA3 but did not require DsbA1/2 even though these oxidoreductases could oxidise the protein. This is the first indication that DsbA3 acts as an oxidoreductase in vivo and that multiple oxidoreductases may be involved in oxidising the one target in N. meningitidis. In conclusion, LptA is stabilised by disulphide bonds within the protein. This effect was more pronounced when neisserial LptA was expressed in E. coli than in N. meningitidis and may reflect that other factors in the neisserial periplasm have a role in LptA stability.

Substrate Specificity of the Oxidoreductase ERp57 Is Determined Primarily by Its Interaction with Calnexin and Calreticulin

The formation of disulfides within proteins entering the secretory pathway is catalyzed by the protein disulfide isomerase family of endoplasmic reticulum localized oxidoreductases. One such enzyme, ERp57, is thought to catalyze the isomerization of non-native disulfide bonds formed in glycoproteins with unstructured disulfide-rich domains. Here we investigated the mechanism underlying ERp57 specificity toward glycoprotein substrates and the interdependence of ERp57 and the calnexin cycle for their correct folding. Our results clearly show that ERp57 must be physically associated with the calnexin cycle to catalyze isomerization reactions with most of its substrates. In addition, some glycoproteins only require ERp57 for correct disulfide formation if they enter the calnexin cycle. Hence, the specificity of ER oxidoreductases is not only determined by the physical association of enzyme and substrate but also by accessory factors, such as calnexin and calreticulin in the case of ERp57. These conclusions suggest that the calnexin cycle has evolved with a specialized oxidoreductase to facilitate native disulfide formation in complex glycoproteins.

Systems-based design of bi-ligand inhibitors of oxidoreductases: filling the chemical proteomic toolbox.

Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidoreductase gene family into structural subfamilies termed pharmacofamilies, which share pharmacophore features in their cofactor binding sites. Then we identify a ligand for this site and use NMR-based binding site mapping (NMR SOLVE) to determine where to extend a combinatorial library, such that diversity elements are directed into the adjacent substrate site. The cofactor mimic is reused in the library in a manner that parallels the reuse of cofactor domains in the oxidoreductase gene family. A library designed in this manner yielded specific inhibitors for multiple oxidoreductases.