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...
Expression of a class V chitinase gene (At4g19810, AtChiC) in Arabidopsis thaliana was examined by quantitative real-time PCR and by analyzing microarray data available at Genevestigator. The gene expression was induced by the plant stress-related hormones abscisic acid (ABA) and jasmonic acid (JA) and by the stress resulting from the elicitor flagellin, NaCl, and osmosis. The recombinant AtChiC protein was produced in E. coli, purified, and characterized with respect to the structure and function. The recombinant AtChiC hydrolyzed N-acetylglucosamine oligomers producing dimers from the non-reducing end of the substrates. The crystal structure of AtChiC was determined by the molecular replacement method at 2.0 Å resolution. AtChiC was found to adopt an (β/α)(8) fold with a small insertion domain composed of an α-helix and a five-stranded β-sheet. From docking simulation of AtChiC with pentameric substrate, the amino acid residues responsible for substrate binding were found to be well conserved when compared with those of the class V chitinase from Nicotiana tabacum (NtChiV). All of the structural and functional properties of AtChiC are quite similar to those obtained for NtChiV, and seem to be common to class V chitinases from higher plants.
In the gauge-Higgs unification scenario the Higgs field is unified with gauge fields in higher dimensional gauge theory. The 4D Higgs field H(x) corresponds to 4D fluctuations of the Aharonov-Bohm phase (Wilson line phase) θH in the extra-dimension. An SO(5) × U (1) gauge-Higgs unification model in the Randall-Sundrum warped spacetime with top and bottom quarks is presented. Gauge couplings of the top quark multiplet induce electroweak symmetry breaking by the Hosotani mechanism. The effective potential V eff (θH) is found to be minimized at θH = 1 2 π and the Higgs mass is predicted around 50 GeV. The ZZH and W W H couplings vanish at θH = 1 2 π so that the LEP2 bound for the Higgs mass is evaded. The result is summarized in the effective interactions forθH(x) = θH + H(x)/fH.
Chitinase-A (BcChi-A) was purified from a moss, Bryum coronatum, by several steps of column chromatography. The purified BcChi-A was found to be a molecular mass of 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.5. A cDNA encoding BcChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1012 nucleotides and encoded an open reading frame of 228 amino acid residues. The predicted mature BcChi-A consists of 205 amino acid residues and has a molecular weight of 22,654. Sequence analysis indicated that BcChi-A is glycoside hydrolase family-19 (GH19) chitinase lacking loops I, II, IV and V, and a C-terminal loop, which are present in the catalytic domain of plant class I and II chitinases. BcChi-A is a compact chitinase that has the fewest loop regions of the GH19 chitinases. Enzymatic experiments using chitooligosaccharides showed that BcChi-A has higher activity toward shorter substrates than class II enzymes. This characteristic is likely due to the loss of the loop regions that are located at the end of the substrate-binding cleft and would be involved in substrate binding of class II enzymes. This is the first report of a chitinase from mosses, nonvascular plants.
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