Intermediate Trapping on a Mutant Retaining α-Galactosyltransferase Identifies an Unexpected Aspartate Residue *

Lipopolysaccharyl-alpha-1,4-galactosyltransferase C (LgtC), a glycosyltransferase family 8 alpha-1,4-galactosyltransferase from Neisseria meningitidis, catalyzes the transfer of galactose from UDP galactose to terminal lactose-containing acceptor sugars with net retention of anomeric configuration. To investigate the potential role of discrete nucleophilic catalysis suggested by the double displacement mechanism generally proposed for retaining glycosyltransferases, the side chain amide of Gln-189, which is suitably positioned to act as the catalytic nucleophile of LgtC, was substituted with the more nucleophilic carboxylate-containing side chain of glutamate in the hope of accumulating a glycosyl-enzyme intermediate. The resulting mutant was subjected to kinetic, mass spectrometric, and x-ray crystallographic analysis. Although the K(m) for UDP-galactose is not significantly altered, the k(cat) was reduced to 3% that of the wild type enzyme. Electrospray mass spectrometric analysis revealed that a steady state population of the Q189E variant contains a covalently bound galactosyl moiety. Liquid chromatographic/mass spectrometric analysis of fragmented proteolytic digests identified the site of labeling not as Glu-189 but, surprisingly, as the sequentially adjacent Asp-190. However, the side chain carboxylate of Asp-190 is located 8.9 A away from the donor substrate in the available crystal structure. Kinetic analysis of a D190N mutant at this position revealed a k(cat) value 3000-fold lower than that of the wild type enzyme. A 2.6-A crystal structure of the Q189E mutant with bound uridine 5'-diphospho-2-deoxy-2-fluoro-alpha-d-galactopyranose revealed no significant perturbation of the mode of donor sugar binding nor of active site configuration. This is the first trapping of an intermediate in the active site of a retaining glycosyltransferase and, although not conclusive, implicates Asp-190 as an alternative candidate catalytic nucleophile, thereby rekindling a longstanding mechanistic debate

Problems and Pitfalls of Identifying Remyelination in Multiple Sclerosis.

Regenerative medicines that promote remyelination in multiple sclerosis (MS) are making the transition from laboratory to clinical trials. While animal models provide the experimental flexibility to analyze mechanisms of remyelination, here we discuss the challenges in understanding where and how remyelination occurs in MS.The authors acknowledge the following support: The UK Multiple Sclerosis Society (RTK, CZ, RJMF), The Adelson Medical Research Foundation (DSR, DEB, RJMF), Intramural Research Program of NINDS/NIH (DSR), European Research Council (ERC) under the European Union Horizon 2020 Re- search and Innovation Program (RTK), The Lister Institute (RTK), and a core support grant from the Wellcome and MRC to the Wellcome-Medical Research Council Cambridge Stem Cell Institute (RTK, RJMF)

A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases

Problems and Pitfalls of Identifying Remyelination in Multiple Sclerosis

The authors acknowledge the following support: The UK Multiple Sclerosis Society (MS50 to R.T.K., C.Z., and R.J.M.F.), The Adelson Medical Research Foundation (D.S.R., D.E.B., and R.J.M.F.), Intramural Research Program of NINDS/NIH (D.S.R.), European Research Council (ERC) under the European Union Horizon 2020 Research and Innovation Program (771411 to R.T.K.), The Lister Institute (R.T.K.), and a core support grant from the Wellcome and MRC to the Wellcome-Medical Research Council Cambridge Stem Cell Institute (203151/Z/16/Z to R.T.K. and R.J.M.F.). Publisher Copyright: © 2020 Elsevier Inc.Regenerative medicines that promote remyelination in multiple sclerosis (MS) are making the transition from laboratory to clinical trials. While animal models provide the experimental flexibility to analyze mechanisms of remyelination, here we discuss the challenges in understanding where and how remyelination occurs in MS.Peer reviewe

Mechanisms and engineering of glycosyltransferases

In order to gain insight into the natural evolution of enzyme mechanism and to increase the utility of a class of sugar modifying enzymes known as glycosyltransferases, several representative enzymes were subjected to mechanistic and engineering studies. The chemical and kinetic mechanisms of the GT-A fold inverting sialyltransferase Cst II were investigated by detailed kinetic analysis, protein X-ray crystallography and mutagenesis. This enzyme catalyzes the general SN[subscript]2-like direct displacement mechanism used by all inverting glycosyltransferases. However, the chemical strategies utilized to facilitate reaction are more akin to those of the GT-B fold enzymes, indicating a convergence in mechanism between these two clans of enzymes. By analogy to retaining glycosidases, retaining glycosyltransferases had been thought to use a double displacement mechanism involving an enzymatic nucleophile. However, a comparison of the X-ray crystal structures of multiple retaining glycosyltransferases indicates a complete lack of conserved structural architecture in the region that would be occupied by this critical catalytic residue. This lack of conserved architecture, precedence for cationic enzymatic mechanisms, and the inherent differences in reactivities of glycosyltransferase and glycosidase substrates all support a notion that the majority of retaining glycosyltransferases utilize a mechanism involving the formation of a short-lived ion pair intermediate species. However, a protein engineering approach was used to explore the possibility of nucleophilic catalysis in the retaining galactosyltransferase LgtC. The results of this work led to the first direct observation of a catalytically relevant covalent glycosyl-enzyme intermediate for a retaining glycosyltransferase. It was demonstrated that catalytically active LgtC could be displayed on the surface of M13 bacteriophage as a pIII fusion protein. Further, LgtC phage display was successfully performed in the context of a water-in-oil emulsification procedure that may have significant utility in the development of directed evolution screening approaches. A substrate engineering strategy was developed which allowed the substrate specificity of wild type LgtC to be broadened to allow exclusive formation of α-1,2, α-1,3 or α-1,4 linkages at synthetically useful rates with various alternative acceptor substrates. Finally, it was demonstrated that Cst II and LgtC will utilize alternative donor substrates in the presence of their respective natural nucleotide products.Science, Faculty ofChemistry, Department ofGraduat

Protocol for high-throughput compound screening using flow cytometry in THP-1 cells

Summary: Flow cytometry is a valuable method for analyzing protein expressions at the single cell level but can be difficult to apply to large numbers of samples. This protocol provides instructions to perform a high-throughput small molecule screen using flow cytometry analysis of THP-1 cells, a human monocytic leukemia cell line. We describe a methodology for identifying compounds that regulate PD-L1 surface expression in IFN-γ-stimulated cells, which has been successfully used to screen a collection of ∼200,000 compounds.For complete details on the use and execution of this protocol, please refer to Zavareh et al. (2020)

A novel small-molecule inhibitor of 3-phosphoglycerate dehydrogenase

ABSTRACT Serine metabolism is likely to play a critical role in cancer cell growth. A recent study reports the identification of a novel small-molecule inhibitor of serine synthesis that targets 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme of the serine synthesis pathway, and selectively abrogates the proliferation of PHGDH overexpressing breast cancer cells

Structural and kinetic analysis of substrate binding to the sialyltransferase Cst-II from Campylobacter jejuni.

Peer reviewed: YesNRC publication: Ye