Thioredoxin (Trx), a ubiquitous protein showing disulfide reductase activity, plays critical roles in cellular redox control and oxidative stress response. Trx is a member of the Trx system, comprising Trx, Trx reductase (TrxR), and a cognate reductant (generally reduced nicotinamide adenine dinucleotide phosphate, NADPH). Bacterial Trx1 contains only the Trx-fold domain, in which the active site CXXC motif that is critical for the disulfide reduction activity is located. Bacterial Trx2 contains an N-terminal extension, which forms a zinc-finger domain, including two additional CXXC motifs. The multi-stress resistant bacterium Deinococcus radiodurans encodes both Trx1 (DrTrx1) and Trx2 (DrTrx2), which act as members of the enzymatic antioxidant systems. In this study, we constructed Δdrtrx1 and Δdrtrx2 mutants and examined their survival rates under H₂O₂ treated conditions. Both drtrx1 and drtrx2 genes were induced following H₂O₂ treatment, and the Δdrtrx1 and Δdrtrx2 mutants showed a decrease in resistance toward H₂O₂, compared to the wild-type. Native DrTrx1 and DrTrx2 clearly displayed insulin and DTNB reduction activity, whereas mutant DrTrx1 and DrTrx2, which harbors the substitution of conserved cysteine to serine in its active site CXXC motif, showed almost no reduction activity. Mutations in the zinc binding cysteines did not fully eliminate the reduction activities of DrTrx2. Furthermore, we solved the crystal structure of full-length DrTrx2 at 1.96 Å resolution. The N-terminal zinc-finger domain of Trx2 is thought to be involved in Trx-target interaction and, from our DrTrx2 structure, the orientation of the zinc-finger domain of DrTrx2 and its interdomain interaction, between the Trx-fold domain and the zinc-finger domain, is clearly distinguished from those of the other Trx2 structures.

Terpenoids are the most diverse class of plant primary and specialized metabolites, and trans-prenyltransferases (trans-PTs) are the first branch point to synthesize precursors of various chain lengths for further metabolism. Whereas the catalytic mechanism of the enzyme is known, there is no reliable method for precisely predicting the functions of trans-PTs. With the exponentially increasing number of available trans-PTs genes in public databases, an in silico functional prediction method for this gene family is urgently needed. Here, we present PTS-Pre, a web tool developed on the basis of the “three floors” model, which shows an overall 86% prediction accuracy for 141 experimentally determined trans-PTs. The method was further validated by in vitro enzyme assays for randomly selected trans-PTs. In addition, using this method, we identified nine new GFPPSs from different plants which are beyond the previously reported Brassicaceae clade, suggesting these genes may have occurred via convergent evolution and are more likely lineage-specific. The high accuracy of our blind prediction validated by enzymatic assays suggests that PTS-Pre provides a convenient and reliable method for genome-wide functional prediction of trans-PTs enzymes and will surely benefit the elucidation and metabolic engineering of terpenoid biosynthetic pathways.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related human coronaviruses (hCoVs) regulate host gene expression to facilitate viral replication and evade innate immunity. SARS-CoV-2 nonstructural protein 1 (Nsp1) suppresses host gene expression in part by inhibiting cellular mRNA nuclear export through targeting the mRNA export factor NXF1. However, whether this mechanism is conserved among Nsp1 proteins from diverse hCoVs remains unknown. Here, we demonstrate that Nsp1 proteins from SARS-CoV, SARS-CoV-2, and MERS-CoV, but not those from endemic hCoVs, induce pronounced nuclear accumulation of host poly(A) RNA without altering global transcript levels. Nsp1 proteins from these highly pathogenic hCoVs directly interact with NXF1, whereas endemic hCoV Nsp1 proteins show little or no detectable NXF1 interaction. Structural modeling and mutagenesis identify a conserved N-terminal acidic patch in Nsp1 that contacts the basic surface of the NXF1 RNA recognition motif. Disruption of this interface abolishes the Nsp1-NXF1 interaction and restores host mRNA export while preserving NXF1 interactions with core mRNA export factors. Together, our findings define a conserved structural mechanism by which highly pathogenic hCoV Nsp1 targets NXF1 to inhibit host mRNA export and provide a potential framework for broad-spectrum antiviral intervention.