Katsuyoshi Murata

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Δorf1 and Δorf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild‐type bacteria and non‐glycosylated flagellin from Δorf1 mutant using aspartic N‐peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site‐directed Ser/Ala‐substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix‐assisted laser desorption/ionization time of flight (MALDI‐TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation‐defective mutants retained swimming ability, swarming ability was reduced in the Δorf1, Δorf2 and Ser/Ala‐substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala‐substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule.

Flagellin Glycans from Two Pathovars of Pseudomonas syringae Contain Rhamnose in d and l Configurations in Different Ratios and Modified 4-Amino-4,6-Dideoxyglucose

Flagellins from Pseudomonas syringae pv. glycinea race 4 and Pseudomonas syringae pv. tabaci 6605 have been found to be glycosylated. Glycosylation of flagellin is essential for bacterial virulence and is also involved in the determination of host specificity. Flagellin glycans from both pathovars were characterized, and common sites of glycosylation were identified on six serine residues (positions 143, 164, 176, 183, 193, and 201). The structure of the glycan at serine 201 (S201) of flagellin from each pathovar was determined by sugar composition analysis, mass spectrometry, and (1)H and (13)C nuclear magnetic resonance spectroscopy. These analyses showed that the S201 glycans from both pathovars were composed of a common unique trisaccharide consisting of two rhamnosyl (Rha) residues and one modified 4-amino-4,6-dideoxyglucosyl (Qui4N) residue, beta-D-Quip4N(3-hydroxy-1-oxobutyl)2Me-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap. Furthermore, mass analysis suggests that the glycans on each of the six serine residues are composed of similar trisaccharide units. Determination of the enantiomeric ratio of Rha from the flagellin proteins showed that flagellin from P. syringae pv. tabaci 6605 consisted solely of L-Rha, whereas P. syringae pv. glycinea race 4 flagellin contained both L-Rha and D-Rha at a molar ratio of about 4:1. Taking these findings together with those from our previous study, we conclude that these flagellin glycan structures may be important for the virulence and host specificity of P. syringae.

Active Site Residues and Amino Acid Specificity of the Ubiquitin Carrier Protein-binding RING-H2 Finger Domain

EL5 is a rice ubiquitin-protein isopeptide ligase (E3) containing a RING-H2 finger domain that interacts with Oryza sativa (Os) UBC5b, a rice ubiquitin carrier protein. We introduced point mutations into the EL5 RING-H2 finger so that residues that functionally interact with OsUBC5b could be identified when assayed for ubiquitination activity in vitro. The residue positions were selected based on the results of an EL5 RING-H2 finger/OsUBC5b NMR titration experiment. These RING-H2 finger residues form or are adjacent to a shallow groove that is recognized by OsUBC5b. The E3 activity of EL5 is shown to be dependent on a Trp located at the center of the groove. We classified rice RING fingers according to the type of metal-chelating motif, i.e. RING-H2 or RING-HC, and according to the presence or absence of a conserved EL5-like Trp. We discuss the probable relationship between E3 activity and the conserved Trp.

A study of conformational stability of polypeptide blends by solid state two-dimensional 13C–1H heteronuclear correlation NMR spectroscopy

The intermolecular hydrogen-bond interactions in polyglycine (PG)/poly(L-valine) (PLV) blend with a ratio of 1/1 have been studied through high-speed frequency-switched Lee – Goldburg (FSLG) 13 C– 1 H heteronuclear correlation (HETCOR) NMR experiments, where the PG/PLV blend sample is prepared by adding trifluoroacetic acid (TFA) solution of their polypeptide mixture containing a 2.0 wt/wt% amount of sulfuric acid (H2SO4) to alkaline water. The spectral assignment of the polypeptide blend is made by using multiple proton cross-peaks, which appeared in the HETCOR spectra. Intermolecular correlation peaks between PG(b-sheet) and PLV(b-sheet) appear in the HETCOR spectra with long contact time. From these experimental results, it has been clarified that intermolecular hydrogen-bond interactions ðsCyO ··· H – NrÞ between the PG(b-sheet) and PLV(b-sheet) are formed and thus the PG/PLV blend with high miscibility is formed. q 2003 Elsevier Science Ltd. All rights reserved.

A study of the conformational stability of poly(l-alanine), poly(d-alanine), poly(l-isoleucine), polyglycine and poly(l-valine) and their polypeptide blends in the solid-state by 13C CP/MAS NMR

Abstract 13C CP/MAS NMR and 1H T1ρ experiments on homopolypeptides obtained from d - and l -alanines, l -isoleucine, glycine and l -valine, and on their polypeptide blends have been carried out, in order to elucidate the conformational stability of the polypeptides in the solid-state. These polypeptide blends were prepared by adding trifluoroacetic acid (TFA) solutions of the polypeptides containing a 2.0% (w/w) amount of sulfuric acid (H2SO4) to alkaline water. From these experimental results, it was clarified that the conformations of the polypeptides in their blends are strongly influenced by intermolecular hydrogen bonding interactions which cause their miscibility at the molecular level.

Thermodynamic characterization of OsGID1-gibberellin binding using calorimetry and docking simulations

Gibberellins (GAs) are phytohormones regulating various developmental processes in plants. In rice, the initial GA‐signaling events involve the binding of a GA to the soluble GA receptor protein, GID1. Although X‐ray structures for certain GID1/GA complexes have recently been determined, an examination of the complexes does not fully clarify how GID1s discriminate among different GAs. Herein, we present a study aimed at defining the types of forces important to binding via a combination of isothermal titration calorimetry (ITC) and computational docking studies that employed rice GID1 (OsGID1), OsGID1 mutants, which were designed to have a decreased possible number of hydrogen bonds with bound GA, and GA variants. We find that, in general, GA binding is enthalpically driven and that a hydrogen bond between the phenolic hydroxyl of OsGID1 Tyr134 and the C‐3 hydroxyl of a GA is a defining structural element. A hydrogen‐bond network that involves the C‐6 carboxyl of a GA that directly hydrogen bonds the hydroxyl of Ser198 and indirectly, via a two‐water‐molecule network, the phenolic hydroxyl of Tyr329 and the NH of the amide side‐chain of Asn255 is also important for GA binding. The binding of OsGID1 by GA1 is the most enthalpically driven association found for the biologically active GAs evaluated in this study. This observation might be a consequence of a hydrogen bond formed between the hydroxyl at the C‐13 position of GA1 and the main chain carbonyl of OsGID1 Phe245. Our results demonstrate that by combining ITC experiments and computational methods much can be learned about the thermodynamics of ligand/protein binding. Copyright

Improved expression, purification and crystallization of a putative N-acetyl-γ-glutamyl-phosphate reductase from rice (Oryza sativa)

N-Acetyl-gamma-glutamyl-phosphate reductase (AGPR) catalyzes the third step in an eight-step arginine-biosynthetic pathway that starts with glutamate. This enzyme converts N-acetyl-gamma-glutamyl phosphate to N-acetylglutamate-gamma-semialdehyde by an NADPH-dependent reductive dephosphorylation. AGPR from Oryza sativa (OsAGPR) was expressed in Escherichia coli at 291 K as a soluble fusion protein with an upstream thioredoxin-hexahistidine [Trx-(His)6] extension. OsAGPR(Ala50-Pro366) was purified and crystals were obtained using the sitting-drop vapour-diffusion method at 293 K and diffract X-rays to at least 1.8 A resolution. They belong to the hexagonal space group P6(1), with unit-cell parameters a = 86.11, c = 316.3 A.

A Study of Conformational Stability of Polypeptide Blends by Solid-State NMR Spectroscopy

Abstract In polypeptide blends, the balance of intra- and intermolecular hydrogen bond interactions in two kinds of polypeptide chains play an important role for the conformational stability and the blend miscibility. The observation of the 13 C NMR chemical shifts and relaxation times, and the two-dimensional NMR spectrum leads to a deep understanding of the conformational stability and the miscibility. In this chapter, the most recent research works are introduced.