Shizue Katoh

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.

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.

EL5 is involved in root development as an anti-cell death ubiquitin ligase

Ubiquitin ligase (E3) plays a central role in substrate recognition during ubiquitination, a post-translational modification of proteins. Rice EL5 is an E3 with a RING-H2 finger domain (RFD) and its transcript is upregulated by a chitin elicitor. The EL5-RFD has been intensively studied and demonstrated to exhibit E3 activity. Its three-dimensional structure was determined for the first time in plant E3, and the amino acid residues required for the interaction with the ubiquitin-conjugating enzyme (E2) were identified. Recent analyses revealed that EL5 plays a crucial role as an E3 in the maintenance of cell viability during root development in rice. In this addendum, we report that the EL5-RFD catalyzes polyubiquitination via the Lys48 residue of ubiquitin. We also discuss the possible role of EL5 as an anti-cell death enzyme. We hypothesize that EL5 might be responsible for mediating the degradation of cytotoxic proteins produced in root cells after the actions of phytohormones.

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.