Complete assignment of Ala, Ile, Leu, Met and Val methyl groups of human blood group A and B glycosyltransferases using lanthanide-induced pseudocontact shifts and methyl-methyl NOESY.

Human blood group A and B glycosyltransferases (GTA, GTB) are highly homologous glycosyltransferases. A number of high-resolution crystal structures is available showing that these enzymes convert from an open conformation into a catalytically active closed conformation upon substrate binding. However, the mechanism of glycosyltransfer is still under debate, and the precise nature as well as the time scales of conformational transitions are unknown. NMR offers a variety of experiments to shine more light on these unresolved questions. Therefore, in a first step we have assigned all methyl resonance signals in MILVA labeled samples of GTA and GTB, still a challenging task for 70 kDa homodimeric proteins. Assignments were obtained from methyl-methyl NOESY experiments, and from measurements of lanthanide-induced pseudocontact shifts (PCS) using high resolution crystal structures as templates. PCSs and chemical shift perturbations, induced by substrate analogue binding, suggest that the fully closed state is not adopted in the presence of lanthanide ions.

Substrate Binding Drives Active-Site Closing of Human Blood Group†B Galactosyltransferase as Revealed by Hot-Spot Labeling and NMR Spectroscopy Experiments.

Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between "open", "semiclosed", and "closed" conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at "conformational hot spots" of the enzymes. Therefore, we utilized 15 N side-chain labeling of Trp residues and 13 C-methyl labeling of Met residues to study substrate-induced conformational transitions of GTB. Chemical-shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical-shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200-300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB.

Protein NMR Studies of Substrate Binding to Human Blood Group†A and B Glycosyltransferases.

Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies had shown these enzymes to adopt an open conformation in the absence of substrates. Binding either of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical-shift titrations of uniformly 2 H,15 N-labeled GTA or GTB with UDP affected about 20 % of all crosspeaks in 1 H,15 N TROSY-HSQC spectra, reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical-shift-perturbation experiments with δ1-[13 C]methyl-Ile-labeled samples revealed two Ile residues-Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop-that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY-based relaxation dispersion experiments do not reveal micro- to millisecond timescale motions. Although this study reveals substantial conformational plasticity of GTA and GTB, the matter of how binding of substrates shifts the enzymes into catalytically competent states remains enigmatic.

Food Fingerprinting: LC-ESI-IM-QTOF-Based Identification of Blumeatin as a New Marker Metabolite for the Detection of Origanum majorana Admixtures to O. onites/vulgare.

Oregano (Origanum vulgare and O. onites) is one of the most frequently counterfeited herbs in the world and is diluted with the leaves of a wide variety of plants. In addition to olive leaves, marjoram (O. majorana) is often used for this purpose in order to achieve a higher profit. However, apart from arbutin, no marker metabolites are known to reliably detect marjoram admixtures in oregano batches at low concentrations. In addition, arbutin is relatively widespread in the plant kingdom, which is why it is of great relevance to look for further marker metabolites in order to secure the analysis accordingly. Therefore, the aim of the present study was to use a metabolomics-based approach to identify additional marker metabolites with the aid of an ion mobility mass spectrometry instrument. The focus of the analysis was on the detection of non-polar metabolites, as this study was preceded by nuclear magnetic resonance spectroscopic investigations of the same samples based mainly on the detection of polar analytes. Using the MS-based approach, numerous marjoram specific features could be detected in admixtures of marjoram >10% in oregano. However, only one feature was detectable in admixtures of >5% marjoram. This feature was identified as blumeatin, which belongs to the class of flavonoid compounds. Initially, blumeatin was identified based on MS/MS spectra and collision cross section values using a database search. In addition, the identification of blumeatin was confirmed by a reference standard. Moreover, dried leaves of olive, myrtle, thyme, sage and peppermint, which are also known to be used to adulterate oregano, were measured. Blumeatin could not be detected in these plants, so this substance can be considered as an excellent marker compound for the detection of marjoram admixtures.

Insights into Allosteric Control of Human Blood Group A and B Glycosyltransferases from Dynamic NMR.

Human blood group A and B glycosyltransferases (GTA, GTB) are retaining glycosyltransferases, requiring a catalytic mechanism that conserves the anomeric configuration of the hexopyranose moiety of the donor substrate (UDP-GalNAc, UDP-Gal). Previous studies have shown that GTA and GTB cycle through structurally distinct states during catalysis. Here, we link binding and release of substrates, substrate-analogs, and products to transitions between open, semi-closed, and closed states of the enzymes. Methyl TROSY based titration experiments in combination with zz-exchange experiments uncover dramatic changes of binding kinetics associated with allosteric interactions between donor-type and acceptor-type ligands. Taken together, this highlights how allosteric control of on- and off-rates correlates with conformational changes, driving catalysis to completion.

Recombinant hTRBP and hPACT Modulate hAgo2-Catalyzed siRNA-Mediated Target RNA Cleavage In Vitro.

The human TAR RNA-binding protein (hTRBP) and protein activator of protein kinase R (hPACT) are important players in RNA interference (RNAi). Together with hArgonaute2 (hAgo2) and hDicer they have been reported to form the RISC-loading complex (RLC). Among other functions, hTRBP was suggested to assist the loading of hAgo2 with small interfering RNAs (siRNAs) within the RLC. Although several studies have been conducted to evaluate the specific functions of hTRBP and hPACT in RNAi, exact mechanisms and modes of action are still unknown. Here, we present a biochemical study further evaluating the role of hTRBP and hPACT in hAgo2-loading. We found that both proteins enhance hAgo2-mediated RNA cleavage significantly; even a hAgo2 mutant impaired in siRNA binding shows full cleavage activity in the presence of hTRBP or hPACT. Pre-steady state binding studies reveal that the assembly of wildtype-hAgo2 (wt-hAgo2) and siRNAs remains largely unaffected, whereas the binding of mutant hAgo2-PAZ9 to siRNA is restored by adding either hTRBP or hPACT. We conclude that both proteins assist in positioning the siRNA within hAgo2 to ensure optimal binding and cleavage. Overall, our data indicate that hTRBP and hPACT are part of a regulative system of RNAi that is important for efficient target RNA cleavage.