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RNA oxidation, predominantly through the accumulation of 8-oxo-7,8-dihydroguanosine (8-oxo-rG), represents an important biomarker for cellular oxidative stress. Polynucleotide phosphorylase (PNPase) is a 3′-5′ exoribonuclease that has been shown to preferentially recognize 8-oxo-rG-containing RNA and protect Escherichia coli cells from oxidative stress. However, the impact of 8-oxo-rG on PNPase-mediated RNA degradation has not been studied. Here, we show that the presence of 8-oxo-rG in RNA leads to catalytic stalling of E. coli PNPase through in vitro RNA degradation experiments and electrophoretic analysis. We also link this stalling to the active site of the enzyme through resolution of single-particle cryo-EM structures for PNPase in complex with singly or doubly oxidized RNA oligonucleotides. Following identification of Arg399 as a key residue in recognition of both single and sequential 8-oxo-rG nucleotides, we perform follow-up in vitro analysis to confirm the importance of this residue in 8-oxo-rG-specific PNPase stalling. Finally, we investigate the effects of mutations to active site residues implicated in 8-oxo-rG binding through E. coli cell growth experiments under H2O2-induced oxidative stress. Specifically, Arg399 mutations show significant effects on cell growth under oxidative stress. Overall, we demonstrate that 8-oxo-rG-specific stalling of PNPase is relevant to bacterial survival under oxidative stress and speculate that this enzyme might associate with other cellular factors to mediate this stress. 

Abstract The duality of nitrate is nowhere better exemplified than in human physiology—a detrimental pollutant but also a protective nutrient—particularly as connected to nitric oxide. Aside from limited insights into nitrate uptake and storage, foundational nitrate biology has lagged. Genetically encoded fluorescent biosensors can address this gap with real‐time imaging, but such technologies for mammalian cell applications remain rare. Here, we designed and engineered a biosensor fusing the green fluorescent protein EGFP and the nitrate recognition domain NreA fromStaphylococcus carnosus. Seven rounds of directed evolution and 15 mutations resulted in NitrOFF. NitrOFF has a high degree of allosteric communication between the domains reflected in a turn‐off intensiometric response (K_d≈ 9 µM). This was further reinforced by X‐ray crystal structures of apo and nitrate‐bound NitrOFF, which revealed a large‐scale conformational rearrangement changing the relative positioning of the domains by 68.4°. This dramatic difference was triggered by the formation of a long helix at the engineered linker connecting the two domains, peeling the β7 strand off the EGFP and thus extinguishing the fluorescence upon nitrate binding. Finally, we highlighted the utility of NitrOFF to monitor exogenous nitrate uptake and modulation in the human embryonic kidney (HEK) 293 cell line. 

Abstract The key to type 1 copper (T1Cu) function lies in the fine tuning of the Cu^II/Ireduction potential (E°′_T1Cu) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tuneE°′_T1Cuin azurin over a wide range, these principles are yet to be generalized to other T1Cu‐containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) fromStreptomyces coelicolorto match the highE°′_T1Cuof fungal laccases. Using ultraviolet‐visible absorption and electron paramagnetic resonance spectroscopies, together with X‐ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and correspondingE°′_T1Cu. While minimal and smallE°′_T1Cuincreases are observed in Y229F‐ and S292F‐SLAC, the V290N mutant exhibits a majorE°′_T1Cuincrease. Moreover, the influence of these mutations onE°′_T1Cuis additive, culminating in a triple mutant Y229F/V290N/S292F‐SLAC with the highestE°′_T1Cuof 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (k_cat/K_M) relative to WT‐SLAC.