No abstract

Introducing structural modifications into biomolecules represents a powerful approach to dissect their functions and roles in biological processes. Bacterial polysaccharides, despite their rich structural information and essential roles in bacterium-host interactions and bacterial virulence, have largely been unexplored for in vivo structural modifications. In this study, we demonstrate the incorporation of a panel of monosaccharide analogs into bacterial polysaccharides in a highly homogenous manner via metabolic engineering of a promiscuous sugar nucleotide biosynthetic pathway. In addition, the bioorthorgonal functional groups metabolically incorporated were exploited for cell surface labeling using in vitro selective chemical ligation reactions. In summary, our study presents a general, facile and effective approach for in vivo generation of novel tailor-made bacterial polysaccharides.chemical remodeling ͉ metabolic engineering ͉ sugar nucleotide biosynthesis ͉ fucose

The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, we determined cryo–electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.

Coronavirus 3C-like protease (3CL Pro ) is a highly conserved cysteine protease employing a catalytic dyad for its functions. 3CL Pro is essential to the viral life cycle and, therefore, is an attractive target for developing antiviral agents. However, the detailed catalytic mechanism of coronavirus 3CL Pro remains largely unknown. We took an integrated approach of employing X-ray crystallography, mutational studies, enzyme kinetics study, and inhibitors to gain insights into the mechanism. Such experimental work is supplemented by computational studies, including the prereaction state analysis, the ab initio calculation of the critical catalytic step, and the molecular dynamic simulation of the wild-type and mutant enzymes. Taken together, such studies allowed us to identify a residue pair (Glu-His) and a conserved His as critical for binding; a conserved GSCGS motif as important for the start of catalysis, a partial negative charge cluster (PNCC) formed by Arg-Tyr-Asp as essential for catalysis, and a conserved water molecule mediating the remote interaction between PNCC and catalytic dyad. The data collected and our insights into the detailed mechanism have allowed us to achieve a good understanding of the difference in catalytic efficiency between 3CL Pro from SARS and MERS, conduct mutational studies to improve the catalytic activity by 8-fold, optimize existing inhibitors to improve the potency by 4-fold, and identify a potential allosteric site for inhibitor design. All such results reinforce each other to support the overall catalytic mechanism proposed herein.

O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac4dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac4dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.