Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-GlcNAc transferase using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human O-GlcNAc transferase recognizes the sugar donor and acceptor peptide and employs a novel catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base, as well as an essential lysine. This mechanism appears to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate, and explains the unexpected specificity of a recently reported metabolic O-GlcNAc transferase inhibitor.

Mutations in rumi result in a temperature-sensitive loss of Notch signaling in Drosophila . Drosophila Rumi is a soluble, endoplasmic reticulum-retained protein with a CAP10 domain that functions as a protein O -glucosyltransferase. In human and mouse genomes, three potential Rumi homologues exist: one with a high degree of identity to Drosophila Rumi (52%), and two others with lower degrees of identity but including a CAP10 domain (KDELC1 and KDELC2). Here we show that both mouse and human Rumi, but not KDELC1 or KDELC2, catalyze transfer of glucose from UDP-glucose to an EGF repeat from human factor VII. Similarly, human Rumi, but not KDELC1 or KDELC2, rescues the Notch phenotypes in Drosophila rumi clones. During characterization of the Rumi enzymes, we noted that, in addition to protein O -glucosyltransferase activity, both mammalian and Drosophila Rumi also showed significant protein O -xylosyltransferase activity. Rumi transfers Xyl or glucose to serine 52 in the O -glucose consensus sequence ( ) of factor VII EGF repeat. Surprisingly, the second serine (S53) facilitates transfer of Xyl, but not glucose, to the EGF repeat by Rumi. EGF16 of mouse Notch2, which has a diserine motif in the consensus sequence ( ), is also modified with either O -Xyl or O -glucose glycans in cells. Mutation of the second serine (S590A) causes a loss of O -Xyl but not O -glucose at this site. Altogether, our data establish dual substrate specificity for the glycosyltransferase Rumi and provide evidence that amino acid sequences of the recipient EGF repeat significantly influence which donor substrate (UDP-glucose or UDP-Xyl) is used.

Background:The type III secretion (T3S) chaperone Scc4 modulates Chlamydia RNA polymerase holoenzyme activity and is also required for secretion of the gatekeeper CopN. Results: Interactions between the Scc4 and Scc1 chaperones and CopN are characterized. Conclusion: Scc4 forms a ternary complex with Scc1 and CopN to promote CopN secretion during infection. Significance: Scc4 is an important link between the T3S system and transcription.

An automated method for high-throughput nuclear magnetic resonance (NMR) spectroscopy has been developed using a four-coil Multiplex NMR probe. The probe is constructed with solenoidal microcoils optimized for detection of small volume, mass-limited samples and a flow-through design. Four samples can be simultaneously injected into the Multiplex probe with a robotics liquid handler and then analyzed in rapid succession using a selective excitation experiment. Due to the simultaneous injection of four samples and the reduced analysis time with rapid selective excitation, the analysis rate achieved thus far is as low as 1 sample/34 s for 1D 1H NMR.

Two improved approaches for the rapid analysis of multiple samples using multiplex sample NMR are described. In the first approach, frequency-selective 90 degrees radio frequency pulses and large pulsed field gradients are applied to excite and detect multiple samples in rapid succession. This method is advantageous for samples with relatively long longitudinal (T1) relaxation times. In the second approach, chemical shift imaging is applied to acquire both the spectral and spatial information of multiple samples simultaneously. Chemical shift imaging is more time-consuming than selective excitation; however, it is advantageous for detecting samples with short T1's and for signal averaging. Both approaches demonstrate the potential of multiplex sample NMR for carrying out high-throughput NMR detection.