N,N',N'',N'''-Tetraacetylchitotetraose

Quantification of Protein-Ligand Interactions by Laser Electrospray Mass Spectrometry.[Pubmed:29654537]

J Am Soc Mass Spectrom. 2018 Jul;29(7):1484-1492.

Laser electrospray mass spectrometry (LEMS) measurement of the dissociation constant (K(d)) for hen egg white lysozyme (HEWL) and N,N',N''-triacetylchitotriose (NAG(3)) revealed an apparent K(d) value of 313.2 +/- 25.9 muM for the ligand titration method. Similar measurements for N,N',N'',N'''-tetraacetylchitotetraose (NAG(4)) revealed an apparent K(d) of 249.3 +/- 13.6 muM. An electrospray ionization mass spectrometry (ESI-MS) experiment determined a K(d) value of 9.8 +/- 0.6 muM. In a second LEMS approach, a calibrated measurement was used to determine a K(d) value of 6.8 +/- 1.5 muM for NAG(3). The capture efficiency of LEMS was measured to be 3.6 +/- 1.8% and is defined as the fraction of LEMS sample detected after merging with the ESI plume. When the dilution is factored into the ligand titration measurement, the adjusted K(d) value was 11.3 muM for NAG(3) and 9.0 muM for NAG(4). The calibration method for measuring K(d) developed in this study can be applied to solutions containing unknown analyte concentrations. Graphical Abstract.

Structure, Catalysis, and Inhibition of OfChi-h, the Lepidoptera-exclusive Insect Chitinase.[Pubmed:28053084]

J Biol Chem. 2017 Feb 10;292(6):2080-2088.

Chitinase-h (Chi-h) is of special interest among insect chitinases due to its exclusive distribution in lepidopteran insects and high sequence identity with bacterial and baculovirus homologs. Here OfChi-h, a Chi-h from Ostrinia furnacalis, was investigated. Crystal structures of both OfChi-h and its complex with chitoheptaose ((GlcN)(7)) reveal that OfChi-h possesses a long and asymmetric substrate binding cleft, which is a typical characteristics of a processive exo-chitinase. The structural comparison between OfChi-h and its bacterial homolog SmChiA uncovered two phenylalanine-to-tryptophan site variants in OfChi-h at subsites +2 and possibly -7. The F232W/F396W double mutant endowed SmChiA with higher hydrolytic activities toward insoluble substrates, such as insect cuticle, alpha-chitin, and chitin nanowhisker. An enzymatic assay demonstrated that OfChi-h outperformed OfChtI, an insect endo-chitinase, toward the insoluble substrates, but showed lower activity toward the soluble substrate ethylene glycol chitin. Furthermore, OfChi-h was found to be inhibited by N,N',N''-trimethylglucosamine-N,N',N'',N'''-tetraacetylchitotetraose (TMG-(GlcNAc)(4)), a substrate analog which can be degraded into TMG-(GlcNAc)(1-2) Injection of TMG-(GlcNAc)(4) into 5th-instar O. furnacalis larvae led to severe defects in pupation. This work provides insights into a molting-indispensable insect chitinase that is phylogenetically closer to bacterial chitinases than insect chitinases.

Microbial oligosaccharides differentially induce volatiles and signalling components in Medicago truncatula.[Pubmed:18534640]

Phytochemistry. 2008 Jul;69(10):2029-40.

Plants perceive biotic stimuli by recognising a multitude of different signalling compounds originating from the interacting organisms. Some of these substances represent pathogen-associated molecular patterns, which act as general elicitors of defence reactions. But also beneficial microorganisms like rhizobia take advantage of compounds structurally related to certain elicitors, i.e. Nod-factors, to communicate their presence to the host plant. In a bioassay-based study we aimed to determine to what extent distinct oligosaccharidic signals are able to elicit overlapping responses, including the emission of volatile organic compounds which is mainly considered a typical mode of inducible indirect defence against herbivores. The model legume Medicago truncatula Gaertn. was challenged with pathogen elicitors (beta-(1,3)-beta-(1,6)-glucans and N,N',N'',N'''-tetraacetylchitotetraose) and two Nod-factors, with one of them being able to induce a nodulation response in M. truncatula. Single oligosaccharidic elicitors caused the emission of volatile organic compounds, mainly sesquiterpenoids. The volatile blends detected were quite characteristic for the applied compounds, which could be pinpointed by multivariate statistical methods. As potential mediators of this response, the levels of jasmonic acid and salicylic acid were determined. Strikingly, neither of these phytohormones exhibited changing levels correlating with enhanced volatile emission. All stimuli tested caused an overproduction of reactive oxygen species, whereas nitric oxide accumulation was only effected by elicitors that were equally able to induce volatile emission. Thus, all signalling compounds tested elicited distinct reaction patterns. However, similarities between defence reactions induced by herbivory and pathogen-derived elicitors could be ascertained; but also Nod-factors were able to trigger defence-related reactions.