Structural and Mutational Analyses of Trehalose Synthase from Deinococcus radiodurans Reveal the Interconversion of Maltose-Trehalose Mechanism.

Trehalose synthase (TreS) catalyzes the reversible interconversion of maltose to trehalose, playing a vital role in trehalose production. Understanding the catalytic mechanism of TreS is crucial for optimizing the enzyme activity and enhancing its suitability for industrial applications. Here, we report the crystal structures of both the wild type and the E324D mutant of Deinococcus radiodurans trehalose synthase in complex with the trehalose analogue, validoxylamine A. By employing structure-guided mutagenesis, we identified N253, E320, and E324 as crucial residues within the +1 subsite for isomerase activity. Based on these complex structures, we propose the catalytic mechanism underlying the reversible interconversion of maltose to trehalose. These findings significantly advance our comprehension of the reaction mechanism of TreS.

Crystal structures of Aspergillus oryzae Rib2 deaminase: the functional mechanism involved in riboflavin biosynthesis.

Riboflavin serves as the direct precursor of the FAD/FMN coenzymes and is biosynthesized in most prokaryotes, fungi and plants. Fungal Rib2 possesses a deaminase domain for deamination of pyrimidine in the third step of riboflavin biosynthesis. Here, four high-resolution crystal structures of a Rib2 deaminase from Aspergillus oryzae (AoRib2) are reported which display three distinct occluded, open and complex forms that are involved in substrate binding and catalysis. In addition to the deaminase domain, AoRib2 contains a unique C-terminal segment which is rich in charged residues. Deletion of this unique segment has no effect on either enzyme activity or protein stability. Nevertheless, the C-terminal αF helix preceding the segment plays a role in maintaining protein stability and activity. Unexpectedly, AoRib2 is the first mononucleotide deaminase found to exist as a monomer, perhaps due to the assistance of its unique longer loops (Lß1-ß2, LαB-ß3 and LαC-ß4). These results form the basis for a molecular understanding of riboflavin biosynthesis in fungi and might assist in the development of antibiotics.

Quorum-Sensing Regulator OpaR Directly Represses Seven Protease Genes in Vibrio parahaemolyticus.

Proteases play a key role in numerous bacterial physiological events. Microbial proteases are used in the pharmaceutical industry and in biomedical applications. The genus Vibrio comprises protease-producing bacteria. Proteases transform polypeptides into shorter chains for easier utilization. They also function as a virulence factor in pathogens. The mechanism by which protease genes are regulated in Vibrio parahaemolyticus, an emerging world-wide human pathogen, however, still remains unclear. Quorum sensing is the communication system of bacteria. OpaR is the master quorum-sensing regulator in V. parahaemolyticus. In the present study, quantitative reverse transcriptase-polymerase chain reaction and protease gene promoter-fusion reporter assays revealed that OpaR represses seven protease genes-three metalloprotease genes and four serine protease genes-which are involved in environmental survival and bacterial virulence. Furthermore, the electrophoresis mobility shift assay demonstrated that OpaR is bound directly to the promoter region of each of the seven protease genes. DNase I footprinting identified the sequence of these OpaR-binding sites. ChIP-seq analyses revealed 435 and 835 OpaR-binding sites in the late-log and stationary phases, respectively. These OpaR-binding sequences indicated a conserved OpaR-binding motif: TATTGATAAAATTATCAATA. These results advance our understanding of the protease regulation system in V. parahaemolyticus. This study is the first to reveal the OpaR motif within V. parahaemolyticus in vivo, using ChIP-seq, and to provide a database for OpaR direct regulon.

Structural, Biophysical, and Biochemical Elucidation of the SARS-CoV-2 Nonstructural Protein 3 Macro Domain.

The pandemic outbreak of a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has threatened the global public health and economy since late December 2019. SARS-CoV-2 encodes the conserved macro domain within nonstructural protein 3, which may reverse cellular ADP-ribosylation and potentially cut the signal of a viral infection in the cell. Herein, we report that the SARS-CoV-2 macro domain was examined as a poly-ADP-ribose (ADPR) binding module and possessed mono-ADPR cleavage enzyme activity. After confirming the ADPR binding ability via a biophysical approach, the X-ray crystal structure of the SARS-CoV-2 macro domain was determined and structurally compared with those of other viruses. This study provides structural, biophysical, and biochemical bases to further evaluate the role of the SARS-CoV-2 macro domain in the host response via ADP-ribose binding but also as a potential target for drug design against COVID-19.

OpaR and RpoS are positive regulators of a virulence factor PrtA in Vibrio parahaemolyticus.

PrtA is an extracellular serine protease of Vibrio parahaemolyticus and has haemolytic and cytotoxic activities. Many extracellular proteases have been shown to be required for nutrient intake and the infection mechanism of vibrios. In this study, we report that OpaR, a quorum sensing regulator, and RpoS, a general stress response regulator, play important roles in the PrtA regulation pathway. Extracellular protease activity was highest during the late-log growth of Vibrio parahaemolyticus no.93 (VP93). The absence of PrtA distinctly decreased the extracellular protease activity. Deletion of opaR or rpoS alone reduced PrtA-specific activity of VP93. Quantitative reverse-transcriptase PCR and Western blot analysis suggested that OpaR and RpoS promote PrtA expression at the transcriptional level and affect the amount of extracellular PrtA. A luciferase assay revealed that OpaR regulates prtA on the prtA promoter region. Electrophoretic mobility shift assays indicated that the purified His-OpaR was able to bind specifically to two sequences (PrtA-1 and PrtA-2) of the prtA promoter region. Footprinting analysis showed that OpaR regulates prtA by binding to the promoter region of prtA at positions -269 to -246 and -88 to -68 from the prtA translational start site. Together, the results suggest that PrtA was upregulated by two global regulators, OpaR and RpoS.