Structural insights into Xanthomonas campestris pv. campestris NAD+ biosynthesis via the NAM salvage pathway.

Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) via the nicotinamide (NAM) salvage pathway. While the structural biochemistry of eukaryote NAMPT has been well studied, the catalysis mechanism of prokaryote NAMPT at the molecular level remains largely unclear. Here, we demonstrated the NAMPT-mediated salvage pathway is functional in the Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) for the synthesis of NAD+, and the enzyme activity of NAMPT in this bacterium is significantly higher than that of human NAMPT in vitro. Our structural analyses of Xcc NAMPT, both in isolation and in complex with either the substrate NAM or the product nicotinamide mononucleotide (NMN), uncovered significant details of substrate recognition. Specifically, we revealed the presence of a NAM binding tunnel that connects the active site, and this tunnel is essential for both catalysis and inhibitor binding. We further demonstrated that NAM binding in the tunnel has a positive cooperative effect with NAM binding in the catalytic site. Additionally, we discovered that phosphorylation of the His residue at position 229 enhances the substrate binding affinity of Xcc NAMPT and is important for its catalytic activity. This work reveals the importance of NAMPT in bacterial NAD+ synthesis and provides insights into the substrate recognition and the catalytic mechanism of bacterial type II phosphoribosyltransferases.

Structural and biochemical basis of FLS2-mediated signal activation and transduction in rice.

The receptor-like kinase FLAGELLIN-SENSITIVE 2 (FLS2) functions as a bacterial flagellin receptor localized on the cell membrane of plants. In Arabidopsis, the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) cooperates with FLS2 to detect the flagellin epitope flg22, resulting in formation of a signaling complex that triggers plant defense responses. However, the co-receptor responsible for recognizing and signaling the flg22 epitope in rice remains to be determined, and the precise structural mechanism underlying FLS2-mediated signal activation and transduction has not been clarified. This study presents the structural characterization of a kinase-dead mutant of the intracellular kinase domain of OsFLS2 (OsFLS2-KDD1013A) in complex with ATP or ADP, resolved at resolutions of 1.98 Å and 2.09 Å, respectively. Structural analysis revealed that OsFLS2 can adopt an active conformation in the absence of phosphorylation, although it exhibits only weak basal catalytic activity for autophosphorylation. Subsequent investigations demonstrated that OsSERK2 effectively phosphorylates OsFLS2, which reciprocally phosphorylates OsSERK2, leading to complete activation of OsSERK2 and rapid phosphorylation of the downstream substrate receptor-like cytoplasmic kinases OsRLCK176 and OsRLCK185. Through mass spectrometry experiments, we successfully identified critical autophosphorylation sites on OsSERK2, as well as sites transphosphorylated by OsFLS2. Furthermore, we demonstrated the interaction between OsSERK2 and OsFLS2, which is enhanced in the presence of flg22. Genetic evidence suggests that OsRLCK176 and OsRLCK185 may function downstream of the OsFLS2-mediated signaling pathway. Our study reveals the molecular mechanism by which OsFLS2 mediates signal transduction pathways in rice and provides a valuable example for understanding RLK-mediated signaling pathways in plants.

Crystal structure of transcription factor TGA7 from Arabidopsis.

TGA family of transcription factors play important roles in the systemic acquired resistance (SAR) in plants. In SAR, TGA7 binds to the activation sequence-1 (as-1) in the promoter region of SAR related genes and regulates their expressions in an NPR1 dependent manner. Despite its important roles in plant immunity, the molecular mechanism for DNA binding of TGA7 remains unclear. In the present work, we resolved the crystal structure of TGA7 dimers at a resolution of 2.06 Å, in which each monomer binds one molecule of palmitate. Further biochemical studies revealed that TGA7 specifically binds to the TGACG boxes of as-1 DNA in the form of homodimers, and it has specific requirements for the relative spacing and orientation of the two TGACG boxes. Moreover, we built a TGA7-DNA complex model and confirmed by site-directed mutagenesis that amino acid residue R109 in the DNA binding domain (DBD) of TGA7 is a key residue responsible for DNA recognition. Our work offers a good example for structural and functional studies of TGA proteins, and provides key clues to understand the DNA binding mechanism of TGA proteins in the SAR.

Structural Insights into the Catalytic Mechanism and Ubiquitin Recognition of USP34.

Ubiquitination, an important posttranslational modification, participates in virtually all aspects of cellular functions and is reversed by deubiquitinating enzymes (DUBs). Ubiquitin-specific protease 34 (USP34) plays an essential role in cancer, neurodegenerative diseases, and osteogenesis. Despite its functional importance, how USP34 recognizes ubiquitin and catalyzes deubiquitination remains structurally uncharacterized. Here, we report the crystal structures of the USP34 catalytic domain in free state and after binding with ubiquitin. In the free state, USP34 adopts an inactive conformation, which contains a misaligned catalytic histidine in the triad. Comparison of USP34 structures before and after ubiquitin binding reveals a structural basis for ubiquitin recognition and elucidates a mechanism by which the catalytic triad is realigned. Transition from an open inactive state to a relatively closed active state is coupled to a process by which the "fingertips" of USP34 intimately grip ubiquitin, and this has not been reported before. Our structural and biochemical analyses provide important insights into the catalytic mechanism and ubiquitin recognition of USP34.

Crystal structure of the acyl acid amido synthetase GH3-8 from Oryza sativa.

The Gretchen Hagen 3 (GH3) family of acyl acid amido synthetases regulate the levels and activities of plant hormones containing carboxyl groups, thereby modulating diverse physiological responses. While structure-function relationships have been elucidated for dicotyledonous GH3s, the catalytic mechanism of monocotyledonous GH3 remains elusive. Rice (Oryza sativa) is a representative monocot, and its yield is controlled by the natural growth hormone IAA (indole-3-acetic acid). OsGH3-8 is a model GH3 enzyme that conjugates excess IAA to amino acids in an ATP-dependent manner, ensuring auxin homeostasis and regulating disease resistance, growth and development. Here, we report the crystal structure of OsGH3-8 protein in complex with AMP to uncover the molecular and structural basis for the activity of monocotyledonous GH3-8. Structural and sequence comparisons with other GH3 proteins reveal that the AMP/ATP binding sites are highly conserved. Molecular docking studies with IAA, the GH3-inhibitor Adenosine-5'-[2-(1H-indol-3-yl)ethyl]phosphate (AIEP), and Aspartate provide important information for substrate binding and selectivity of OsGH3-8. Moreover, the observation that AIEP nearly occupies the entire binding site for AMP, IAA and amino acid, offers a ready explanation for the inhibitory effect of AIEP. Taken together, the present study provides vital insights into the molecular mechanisms of monocot GH3 function, and will help to shape the future designs of effective inhibitors.

Effect of Circadian Clock and Light-Dark Cycles in Onchidium reevesii: Possible Implications for Long-Term Memory.

The sea slug Onchidium reevesii inhabits the intertidal zone, which is characterized by a changeable environment. Although the circadian modulation of long-term memory (LTM) is well documented, the interaction of the circadian clock with light-dark masking in LTM of intertidal animals is not well understood. We characterized the LTM of Onchidium and tested the expression levels of related genes under a light-dark (LD) cycle and constant darkness (i.e., dark-dark, or DD) cycle. Results indicated that both learning behavior and LTM show differences between circadian time (CT) 10 and zeitgeber time (ZT) 10. In LD, the cry1 gene expressed irregularly, and per2 expression displayed a daily pattern and a peak expression level at ZT 18. OnCREB1 (only in LD conditions) and per2 transcripts cycled in phase with each other. In DD, the cry1 gene had its peak expression at CT 10, and per2 expressed its peak level at CT 18. OnCREB1 had two peak expression levels at ZT 10 or ZT 18 which correspond to the time node of peaks in cry1 and per2, respectively. The obtained results provide an LTM pattern that is different from other model species of the intertidal zone. We conclude that the daily transcriptional oscillations of Onchidium for LTM were affected by circadian rhythms and LD cycle masking.

Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii.

Real-time quantitative reverse transcription-PCR (qRT-PCR) is an undeniably effective tool for measuring levels of gene expression, but the accuracy and reliability of the statistical data obtained depend mainly on the basal expression of selected housekeeping genes in many samples. To date, there have been few analyses of stable housekeeping genes in Onchidium reevesii under salinity stress and injury. In this study, the gene expression stabilities of seven commonly used housekeeping genes, CYC, RPL28S, ACTB, TUBB, EF1a, Ubiq and 18S RNA, were investigated using BestKeeper, geNorm, NormFinder and RefFinfer. Although the results of the four programs varied to some extent, in general, RPL28S, TUBB, ACTB and EF1a were ranked highly. ACTB and TUBB were found to be the most stable housekeeping genes under salinity stress, and EF1a plus TUBB was the most stable combination under injury stress. When analysing target gene expression in different tissues, RPL28S or EF1a should be selected as the reference gene according to the level of target gene expression. Under extreme environmental stress (salinity) conditions, ACTB (0 ppt, 5 ppt, 15 ppt, 25 ppt) and TUBB (35 ppt) are reasonable reference gene choices when expression stability and abundance are considered. Under conditions of 15 ppt salinity and injury stress, our results showed that the best two-gene combination was TUBB plus EF1a. Therefore, we suggest that RPL28S, ACTB and TUBB are suitable reference genes for evaluating mRNA transcript levels. Based on candidate gene expression analysis, the tolerance of O. reevesii to low salinity (low osmotic pressure) is reduced compared to its tolerance to high salinity (high osmotic pressure). These findings will help researchers obtain accurate results in future quantitative gene expression analyses of O. reevesii under other stress conditions.

The Complete Mitochondrial Genome Sequences of the Philomycus bilineatus (Stylommatophora: Philomycidae) and Phylogenetic Analysis.

The mitochondrial genome (mitogenome) can provide information for phylogenetic analyses and evolutionary biology. We first sequenced, annotated, and characterized the mitogenome of Philomycus bilineatus in this study. The complete mitogenome was 14,347 bp in length, containing 13 protein-coding genes (PCGs), 23 transfer RNA genes, two ribosomal RNA genes, and two non-coding regions (A + T-rich region). There were 15 overlap locations and 18 intergenic spacer regions found throughout the mitogenome of P. bilineatus. The A + T content in the mitogenome was 72.11%. All PCGs used a standard ATN as a start codon, with the exception of cytochrome c oxidase 1 (cox1) and ATP synthase F0 subunit 8 (atp8) with TTG and GTG. Additionally, TAA or TAG was identified as the typical stop codon. All transfer RNA (tRNA) genes had a typical clover-leaf structure, except for trnS1 (AGC), trnS2 (TCA), and trnK (TTT). A phylogenetic analysis with another 37 species of gastropods was performed using Bayesian inference, based on the amino acid sequences of 13 mitochondrial PCGs. The results indicated that P. bilineatus shares a close ancestry with Meghimatium bilineatum. It seems more appropriate to reclassify it as Arionoidea rather than Limacoidea, as previously thought. Our research may provide a new meaningful insight into the evolution of P. bilineatus.