The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) dynamically programs cellular physiology to maintain homeostasis and tailor biochemical pathways to meet context-dependant cellular needs. Despite diverse roles for O-GlcNAc, only two enzymes act antagonistically to govern its cycling; O-GlcNAc transferase (OGT) installs the monosaccharide on target proteins and O-GlcNAc hydrolase (OGA) removes it. Recent literature has exposed a network of mechanisms regulating these two enzymes to choreograph global, and target-specific, O-GlcNAc cycling in response to cellular stress and nutrient availability. Herein, we amalgamate these emerging mechanisms from a structural and molecular perspective to explore how the cell exerts fine control to regulate O-GlcNAcylation of diverse proteins in a selective fashion. Papers of particular interest, published within the period of review, have been highlighted as: *of special interest ** of outstanding interest **[3] An important paper in the field. The authors provide unambiguous evidence for mutations in ogt implicated in XLID influencing OGT function. Despite largely normal global O-GlcNAc levels, significant changes in gene transcription are observed. Furthermore, a compensatory mechanism to maintain global O-GlcNAc levels involving OGT-mSin3A-HDAC1 dependant transcriptional repression at the OGA promoter is proposed. *[6] The authors develop a systematic platform for glycosylation sequence characterization and optimization and use this method to characterize OGT sequence preference. This analysis revealed a surprising preference for an aromatic residue at the-4 subsite. *[10] An asparagine ladder in the TPR domains mediates OGT interaction with protein substrates. * [12] New chemical biology tools to identify features of OGT responsible for substrate targeting. This method uncovered a patch of residues lining the inner surface of the N-terminal domain that contribute to interaction with multiple substrates. **[13] A nice structural study defines the effect of XLID-associated OGT mutation L254F as distorting the TPR helix. * [27] mOGT was shown to be catalytically active in vivo and plays an important role in supporting mitochondrial structure and function. **[28] A fundamental advance in discovery of the OGT NLS and the effects of S389 O-GlcNAcylation and interaction with importin α5 for nuclear import. *[35] The authors demonstrate that OGT is enriched in the post-synaptic density of excitatory neuronal synapses whereby it regulates synapse maturation. *[37] Upon glucagon-induced calcium signalling CamKII phosphorylates OGT at S20, which induces O-GlcNAc modification of Ulk proteins through potentiation of AMPKdependant phosphorylation. **[38] Chk1 phosphorylates OGT S20, and this modification regulates OGT localization to the mid-body and regulation of vimentin bridge formation and severing during mitosis. *[39] LSD2 was shown to act as an E3 ligase to ubiquitinylate OGT and thereby facilitate its UPS mediated degradation. **[43] Circad...

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of b-1,2-mannan oligosaccharides. Here, we identify a class of dualactivity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.

Mannosidases are a diverse group of enzymes that are important in the biological processing of mannose-containing polysaccharides and complex glycoconjugates. They are found in 12 of the >160 sequence-based glycosidase families. We discuss evidence that nature has evolved a small set of common mechanisms that unite almost all of these mannosidase families. Broadly, mannosidases (and the closely related rhamnosidases) perform catalysis through just two conformations of the oxocarbenium ion-like transition state: a B2,5 (or enantiomeric 2,5 B) boat and a 3 H4 half-chair. This extends to a new family (GT108) of GDPMan-dependent -1,2mannosyltransferases/phosphorylases that perform mannosyl transfer through a boat conformation as well as some mannosidases that are metalloenzymes and require divalent cations for catalysis. Yet, among this commonality lies diversity. New evidence shows that one unique family (GH99) of mannosidases use an unusual mechanism involving anchimeric assistance via a 1,2-anhydro sugar (epoxide) intermediate.

Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B or H transition-state (TS) conformations. A prominent unassigned catalytic itinerary is that of exo-1,6-α-mannosidases belonging to CAZy family 125. A published complex of Clostridium perfringens GH125 enzyme with a nonhydrolyzable 1,6-α-thiomannoside substrate mimic bound across the active site revealed an undistorted C conformation and provided no insight into the catalytic pathway of this enzyme. We show through a purely computational approach (QM/MM metadynamics) that sulfur-for-oxygen substitution in the glycosidic linkage fundamentally alters the energetically accessible conformational space of a thiomannoside when bound within the GH125 active site. Modeling of the conformational free energy landscape (FEL) of a thioglycoside strongly favors a mechanistically uninformative C conformation within the GH125 enzyme active site, but the FEL of corresponding O-glycoside substrate reveals a preference for a Michaelis complex in an S conformation (consistent with catalysis through a B TS). This prediction was tested experimentally by determination of the 3D X-ray structure of the pseudo-Michaelis complex of an inactive (D220N) variant of C. perfringens GH125 enzyme in complex with 1,6-α-mannobiose. This complex revealed unambiguous distortion of the -1 subsite mannoside to an S conformation, matching that predicted by theory and supporting an S → B → S conformational itinerary for GH125 α-mannosidases. This work highlights the power of the QM/MM approach and identified shortcomings in the use of nonhydrolyzable substrate analogues for conformational analysis of enzyme-bound species.

Penicillin‐binding protein 3 (PBP3) from Pseudomonas aeruginosa is the molecular target of β‐lactam‐based antibiotics. Structures of PBP3 in complexes with azlocillin and cefoperazone, which are in clinical use for the treatment of pseudomonad infections, have been determined to 2.0 Å resolution. Together with data from other complexes, these structures identify a common set of residues involved in the binding of β‐lactams to PBP3. Comparison of wild‐type and an active site mutant (S294A) showed that increased thermal stability of PBP3 following azlocillin binding was entirely due to covalent binding to S294, whereas cefoperazone binding produces some increase in stability without the covalent link. Consistent with this, a third crystal structure was determined in which the hydrolysis product of cefoperazone was noncovalently bound in the active site of PBP3. This is the first structure of a complex between a penicillin‐binding protein and cephalosporic acid and may be important in the design of new noncovalent PBP3 inhibitors.