Summary Ascorbic acid-glutathione (AsA-GSH) cycle represents important antioxidant defense system in planta. Here we utilized Oncidium cytosolic ascorbate peroxidase ( OgCytAPX ) as a model to demonstrate that CytAPX of several plants possess dual catalytic activity of both AsA and GSH, compared with the monocatalytic activity of Arabidopsis APX ( AtCytAPX ). Structural modeling and site-directed mutagenesis identified that three amino acid residues, Pro 63 , Asp 75 , and Tyr 97 , are required for oxidization of GSH in dual substrate catalytic type. Enzyme kinetic study suggested that AsA and GSH active sites are distinctly located in cytosolic APX structure. Isothermal titration calorimetric and UV-visible analysis confirmed that cytosolic APX is a heme-containing protein, which catalyzes glutathione in addition to ascorbate. Biochemical and physiological evidences of transgenic Arabidopsis overexpressing OgCytAPX1 exhibits efficient reactive oxygen species-scavenging activity, salt and heat tolerances, and early flowering, compared with Arabidopsis overexpressing AtCytAPX . Thus results on dual activity CytAPX impose significant advantage on evolutionary adaptive mechanism in planta.

Poly-ADP-ribosylation, a post-translational modification involved in various cellular processes, is well characterized in eukaryotes but thought to be devoid in bacteria. Here, we solve crystal structures of ADP-ribose–bound poly(ADP-ribose)glycohydrolase from the radioresistant bacterium Deinococcus radiodurans (DrPARG), revealing a solvent-accessible 2’-hydroxy group of ADP-ribose, which suggests that DrPARG may possess endo-glycohydrolase activity toward poly-ADP-ribose (PAR). We confirm the existence of PAR in D. radiodurans and show that disruption of DrPARG expression causes accumulation of endogenous PAR and compromises recovery from UV radiation damage. Moreover, endogenous PAR levels in D. radiodurans are elevated after UV irradiation, indicating that PARylation may be involved in resistance to genotoxic stresses. These findings provide structural insights into a bacterial-type PARG and suggest the existence of a prokaryotic PARylation machinery that may be involved in stress responses.

Sulfur is an essential nutrient that can be converted into utilizable metabolic forms to produce sulfur-containing metabolites in plant. Adenosine 5′-phosphosulfate (APS) reductase (APR) plays a vital role in catalyzing the reduction of activated sulfate to sulfite, which requires glutathione. Previous studies have shown that the C-terminal domain of APR acts as a glutathione-dependent reductase. The crystal structure of the C-terminal redox domain of Arabidopsis APR1 (AtAPR1) shows a conserved α/β thioredoxin fold, but not a glutaredoxin fold. Further biochemical studies of the redox domain from AtAPR1 provided evidence to support the structural observation. Collectively, our results provide structural and biochemical information to explain how the thioredoxin fold exerts the glutaredoxin function in APR.

The macro domain is an ADP-ribose (ADPR) binding module, which is considered to act as a sensor to recognize nicotinamide adenine dinucleotide (NAD) metabolites, including poly ADPR (PAR) and other small molecules. The recognition of macro domains with various ligands is important for a variety of biological functions involved in NAD metabolism, including DNA repair, chromatin remodeling, maintenance of genomic stability, and response to viral infection. Nevertheless, how the macro domain binds to moieties with such structural obstacles using a simple cleft remains a puzzle. We systematically investigated the Middle East respiratory syndrome-coronavirus (MERS-CoV) macro domain for its ligand selectivity and binding properties by structural and biophysical approaches. Of interest, NAD, which is considered not to interact with macro domains, was co-crystallized with the MERS-CoV macro domain. Further studies at physiological temperature revealed that NAD has similar binding ability with ADPR because of the accommodation of the thermal-tunable binding pocket. This study provides the biochemical and structural bases of the detailed ligand-binding mode of the MERS-CoV macro domain. In addition, our observation of enhanced binding affinity of the MERS-CoV macro domain to NAD at physiological temperature highlights the need for further study to reveal the biological functions.

In class II transcription activation, the transcription factor normally binds to the promoter near the −35 position and contacts the domain 4 of σ factors (σ4) to activate transcription. However, σ4 of σ70 appears to be poorly folded on its own. Here, by fusing σ4 with the RNA polymerase β‐flap‐tip‐helix, we constructed two σ4 chimera proteins, one from σ70 ()σ470normalc and another from σS ()σ4Snormalc of Klebsiella pneumoniae. The two chimera proteins well folded into a monomeric form with strong binding affinities for −35 element DNA. Determining the crystal structure of σ4Snormalc in complex with −35 element DNA revealed that σ4Snormalc adopts a similar structure as σ4 in the Escherichia coli RNA polymerase σS holoenzyme and recognizes −35 element DNA specifically by several conserved residues from the helix‐turn‐helix motif. By using nuclear magnetic resonance (NMR), σ470normalc was demonstrated to recognize −35 element DNA similar to σ4Snormalc. Carr‐Purcell‐Meiboom‐Gill relaxation dispersion analyses showed that the N‐terminal helix and the β‐flap‐tip‐helix of σ470normalc have a concurrent transient α‐helical structure and DNA binding reduced the slow dynamics on σ470normalc. Finally, only σ470normalc was shown to interact with the response regulator PmrA and its promoter DNA. The chimera proteins are capable of −35 element DNA recognition and can be used for study with transcription factors or other factors that interact with domain 4 of σ factors.