SummaryA 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, D orf1 and D orf2 , exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from D orf1 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 D orf1 , D orf2 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.

A highly useful, visible-light-driven carboxylation of aryl bromides and chlorides with CO was realized using a combination of Pd(OAc) as a carboxylation catalyst and Ir(ppy)(dtbpy)(PF) as a photoredox catalyst. This carboxylation reaction proceeded in high yields under 1 atm of CO with a variety of functionalized aryl bromides and chlorides without the necessity of using stoichiometric metallic reductants.

A visible light driven catalytic cycle for hydrocarboxylation of alkenes with CO was established using a combination of a Rh(i) complex as a carboxylation catalyst and [Ru(bpy)] (bpy = 2,2'- bipyridyl) as a photoredox catalyst. Two key steps, the generation of Rh(i) hydride species and nucleophilic addition of π-benzyl Rh(i) species to CO, were found to be mediated by light.

EL5, a RING-H2 finger protein, is rapidly induced by N-acetylchitooligosaccharides in rice cell. We expressed the EL5 RING-H2 finger domain in Escherichia coli and determined its structure in solution by NMR spectroscopy. The EL5 RING-H2 finger domain consists of twostranded ␤-sheets (␤1, Ala 147 -Phe 149 ; ␤2, Gly 156 -His 158 ), one ␣-helix (Cys 161 -Leu 166 ), and two large N-and C-terminal loops. It is stabilized by two tetrahedrally coordinated zinc ions. This structure is similar to that of other RING finger domains of proteins of known function. From structural analogies, we inferred that the EL5 RING-H2 finger is a binding domain for ubiquitin-conjugating enzyme (E2). The binding site is probably formed by solvent-exposed hydrophobic residues of the N-and C-terminal loops and the ␣-helix. We demonstrated that the fusion protein with EL5-(96 -181) and maltose-binding protein (MBP) was polyubiquitinated by incubation with ubiquitin, ubiquitin-activating enzyme (E1), and a rice E2 protein, OsUBC5b. This supported the idea that the EL5 RING finger domain is essential for ubiquitin-ligase activity of EL5. By NMR titration experiments, we identified residues that are critical for the interaction between the EL5 RING-H2 finger and OsUBC5b. We conclude that the RING-H2 finger domain of EL5 is the E2 binding site of EL5.Upon sensing the invasion of microorganisms, plants evoke a variety of defense reactions, including the synthesis of antimicrobial compounds (phytoalexins) and proteins. Many of these biochemical reactions are based on the activation of defenserelated genes. In some cases, the level of protein accumulation and the rapidity of gene induction in the host plant are correlated to the degree of its disease resistance. Therefore, it might be possible to control disease resistance by modifying the regulatory factors for the expression of defense-related genes.Such regulatory factors could be elements of signal transduction pathways leading from the recognition of invading pathogens to the activation of defense-related genes. Most of the defense responses are reproducible in suspension-cultured cells treated with specific substances called elicitor (1). Chitin fragments (N-acetylchitooligosaccharides) can act as elicitors (2), which induce the transient expression of several "early responsive" genes, such as EL5 (3). EL5 is a RING finger protein, which is structurally related to proteins of the Arabidopsis ATL family. These proteins are characterized by a transmembrane domain (domain I), basic domain (domain II), conserved domain (domain III), and RING-H2 finger domain (domain IV) followed by the C-terminal region with highly diverse amino acid sequences (4). Although some ATL family genes resemble EL5 in being induced in early stages of the defense responses (5), their biochemical function is obscure. Recently, it was shown that the fusion protein of EL5 with maltose-binding protein (MBP) 1 was polyubiquitinated by incubation with ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2). ...

The LysM domain probably binds peptidoglycans, but how it does so has yet to be described. For this report, we measured the thermal stabilities of recombinant LysM domains derived from Pteris ryukyuensis chitinase-A (PrChi-A) and monitored their binding to N-acetylglucosamine oligomers ((GlcNAc) n ) using differential scanning calorimetry, isothermal titration calorimetry, and NMR spectroscopy. We thereby characterized certain of the domains' functional and structural features. We observed that the domains are very resistant to thermal denaturation and that this resistance depends on the presence of disulfide bonds. We also show that the stoichiometry of (GlcNAc) n /LysM domain binding is 1:1. (GlcNAc) 5 titration experiments, monitored by NMR spectroscopy, allowed us to identify the domain residues that are critical for (GlcNAc) 5 binding. The binding site is a shallow groove formed by the N-terminal part of helix 1, the loop between strand 1 and helix 1, the C-terminal part of helix 2, and the loop between helix 2 and strand 2. Furthermore, mutagenesis experiments reiterate the critical involvement of Tyr 72 in (GlcNAc) n /LysM domain binding. Ours is the first report describing the physical structure of a LysM oligosaccharide-binding site based on experimental data.Carbohydrates are often the signals that initiate biochemical processes. For example, as a response to pathogenic attacks, plant defense proteins first recognize and then attack the carbohydrate components of the pathogens' cell walls (1). As these defense proteins bind carbohydrates (2), via their noncatalytic carbohydrate-binding modules, their binding mechanisms are physiologically interesting. Many carbohydrate-binding modules have been structurally and/or functionally characterized (3) and, based on these characteristics, are currently grouped into 49 families in the Carbohydrate-Active Enzyme (CAZy) data base (available on the World Wide Web).The LysM domain, which probably binds chitin, a ␤-(134)-linked N-acetylglucosamine oligosaccharide ((GlcNAc) n ), was originally identified as a component of bacterial lysins. This domain is found in many of the enzymes involved in cell wall degradation and is also present in other proteins that are associated with bacterial cell walls (4 -8). Basal level resistance by plants against certain pathogens also appears to involve the recognition of chitin oligosaccharides and related compounds. Recently, a chitin oligosaccharide elicitor receptor was purified, and it contains a LysM domain within its extracellular domain (9). Additionally, certain symbiotic relationships between leguminous plants and rhizobial bacteria appear to be mediated by LysM domain/chitin oligosaccharide interactions. Recent studies have identified a class of proteins that recognize Nod factors, which are the lipochitin oligosaccharide signal molecules secreted by symbiotic bacteria (10 -12). These plant proteins are members of a serine/threonine receptor kinase family and contain extracellular LysM domains. After reviewing the availab...

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.The RING finger motif, found in many functionally distinct proteins, was first identified as the protein product of the human gene RING1 (Really Interesting New Gene 1) (1). The RING finger motif is defined by the consensus sequence Cys-X 2 -Cys-X 9 -39 -Cys-X 1-3 -His-X 2-3 -(Cys/ His)-X 2 -Cys-X 4 -48 -Cys-X 2 -Cys, where X is any amino acid and the number of X residues varies in different fingers. Two types of RING finger motifs are distinguished by a cysteine (RING-HC) or histidine (RING-H2) as the fifth metal-chelating residue. A RING finger typically binds two zinc atoms, with its Cys and/or His side chains in a unique "cross-brace" arrangement. The nearly invariant spacing between the second and third pairs of Cys/His residues probably conserves the distance between the two metal-chelating sites (2). RING fingers are commonly found in proteins that are involved in cell growth and differentiation (3). Some RING fingers may be required for protein association, e.g. homo-or heterodimerization, whereas others are needed for ubiquitination (4 -6). The ubiquitination product is an isopeptide bond between the C-terminal carboxyl of ubiquitin (Gly 76 ) and a substrate lysine ⑀-amino group. Ubiquitination requires three sequential enzymatic reactions: (i) a ubiquitin-activating enzyme (E1) 2 forms a thiol ester between one of its cysteines and the ubiquitin Gly 76 carboxyl; (ii) then a conjugating enzyme (ubiquitin carrier protein (E2)) transiently carries the ubiquitin (as a thiol ester); and (iii) a ubiquitin-protein isopeptide ligase (E3) helps to transfer the activated ubiquitin from E2 to the substrate Lys. Generally, eukaryotic cells contain a single type of E1, multiple types of E2, and many different E3 enzymes. Efficient and targeted ubiquitination depends on E3. All known E3 enzymes have one of two E2-binding domains: the RING finger domain or the HECT domain (7). The three-dimensional structures of E3-type RING fingers have been determined by NMR spectroscopy (8 -13) or x-ray diffraction (14 -16). These structures all have a groove formed by the first zincbinding loop (N-loop; Cys 134 -Cys 137 of the...

Selective oxygenation of sulfides to sulfones and sulfoxides was demonstrated by photocatalysis of decavanadate using O2 in methyl ethyl ketone.

This work involved a detailed investigation into the infrared vibrational spectra of ruthenium polypyridyl complexes, specifically heteroleptic [Ru(bpy)2(bpm)](2+) (bpy = 2,2'-bipyridine and bpm = 2,2'-bipyrimidine) and homoleptic [Ru(bpy)3](2+), in the excited triplet state. Transient spectra were acquired 500 ps after photoexcitation, corresponding to the vibrational ground state of the excited triplet state, using time-resolved infrared spectroscopy. We assigned the observed bands to specific ligands in [Ru(bpy)2(bpm)](2+) based on the results of deuterium substitution and identified the corresponding normal vibrational modes using quantum-chemical calculations. Through this process, the more complex vibrational bands of [Ru(bpy)3](2+) were assigned to normal vibrational modes. The results are in good agreement with the model in which excited electrons are localized on a single ligand. We also found that the vibrational bands of both complexes associated with the ligands on which electrons are little localized appear at approximately 1317 and 1608 cm(-1). These assignments should allow the study of the reaction dynamics of various photofunctional systems including ruthenium polypyridyl complexes.