The Diels-Alder [4 + 2] cycloaddition reaction is one of the most powerful and elegant organic synthesis methods for forming 6-membered molecules and has been known for nearly a century. However, whether and how enzymes catalyze this type of reaction is still not completely clear. Here we focus on PyrI4, an enzyme found in the biosynthetic pathway of pyrroindomycins where it catalyzes the formation of a spiro-conjugate via an enzyme-dependent exo-selective [4 + 2] cycloaddition reaction. We report the crystal structures of PyrI4 alone and in complex with its product. Comparative analysis of these structures, combined with biochemical analysis, lead us to propose a unique trapping mechanism whereby the lid-like action of the N-terminal tail imposes conformational constraints on the β barrel catalytic core, which enhances the proximity and polarization effects of reactive groups (1,3-diene and alkene) to drive cyclization in a regio- and stereo-specific manner. This work represents an important step toward the wider application of enzyme-catalyzed [4 + 2] cyclization for synthetic purposes.

Optineurin is an important autophagy receptor involved in several selective autophagy processes, during which its function is regulated by TBK1. Mutations of optineurin and TBK1 are both associated with neurodegenerative diseases. However, the mechanistic basis underlying the specific interaction between optineurin and TBK1 is still elusive. Here we determine the crystal structures of optineurin/TBK1 complex and the related NAP1/TBK1 complex, uncovering the detailed molecular mechanism governing the optineurin and TBK1 interaction, and revealing a general binding mode between TBK1 and its associated adaptor proteins. In addition, we demonstrate that the glaucoma-associated optineurin E50K mutation not only enhances the interaction between optineurin and TBK1 but also alters the oligomeric state of optineurin, and the ALS-related TBK1 E696K mutation specifically disrupts the optineurin/TBK1 complex formation but has little effect on the NAP1/TBK1 complex. Thus, our study provides mechanistic insights into those currently known disease-causing optineurin and TBK1 mutations found in patients.

Background Only few pathogens that cause lower respiratory tract infections (LRTIs) can be identified due to limitations of traditional microbiological methods and the complexity of the oropharyngeal normal flora. Metagenomic next-generation sequencing (mNGS) has the potential to solve this problem. Methods This prospective observational study sequentially enrolled 93 patients with LRTI and 69 patients without LRTI who visited Peking University People’s Hospital in 2019. Pathogens in bronchoalveolar lavage fluid (BALF) specimens were detected using mNGS (DNA and RNA) and traditional microbiological assays. Human transcriptomes were compared between LRTI and non-LRTI, bacterial and viral LRTI, and tuberculosis and nontuberculosis groups. Results Among 93 patients with LRTI, 20%, 35%, and 65% of cases were detected as definite or probable pathogens by culture, all microbiological tests, and mNGS, respectively. Our in-house BALF mNGS platform had an approximately 2-working-day turnaround time and detected more viruses and fungi than the other methods. Taking the composite reference standard as a gold standard, it had a sensitivity of 66.7%, specificity of 75.4%, positive-predictive value of 78.5%, and negative-predictive value of 62.7%. LRTI-, viral LRTI–, and tuberculosis-related differentially expressed genes were respectively related to immunity responses to infection, viral transcription and response to interferon-γ pathways, and perforin 1 and T-cell receptor B variable 9. Conclusions Metagenomic DNA and RNA-seq can identify a wide range of LRTI pathogens, with improved sensitivity for viruses and fungi. Our in-host platform is likely feasible in the clinic. Host transcriptome data are expected to be useful for the diagnosis of LRTIs.