Biochemical Characterization Of A Lipodisaccharide-forming Nod Factor Hydrolase Of Medicago Truncatula

Establishment of symbiosis between legumes and rhizobia depends on rhizobialnodulation signals [Nod factors (NFs); lipochitooligosaccharides]. In the rhizosphereof Medicago truncatula, a lipodisaccharide-forming NF hydrolase plays a key role inthe rapid inactivation of NFs of Sinorhizobium meliloti. Various chitinases possess theability to hydrolyze NFs. In this thesis, a symbiosis related chitinase of M. truncatula(MtChit5) was characterized. MtChit5is a predicted class V chitinase belonging toglycoside hydrolase family18(GH18). To answer the question whether MtChit5isthe lipodisaccharide-forming NF hydrolase, the nodule-induced MtChit5gene wascloned and expressed in Escherichia coli BL21(DE3). For comparison, pointmutations [MtChit5(D148A) and MtChit5(E152A)] and homologous proteins ofMtChit5[i.e. a putative lectin of M. truncatula (MtCRA) and two class V chitinasesof Arabidopsis thaliana (AtChiC) and Nicotiana tabacum (NtChiV)] were also clonedand expressed in E. coli. It was found that MtChit5specifically hydrolyzes NFs of S.meliloti into lipodisaccharides, whereas related molecules (chitooligosaccharides) were not cleaved. AtChiC and NtChiV efficiently cleaved chitooligosaccharides,whereas the point mutated proteins MtChit5(D148A) and MtChit5(E152A) wereinactive. MtCRA also displayed no activity. In contrast to MtChit5, tetrameric NFs ofS. meliloti were not hydrolyzed by AtChiC or NtChiV. MtChit5showedMichaelis-Menten constant (Km) values for tested NFs in the micromolarconcentration range. The Kmvalues of AtChiC or NtChiV for the pentamericNodSm-V(C16:2, S) were considerably higher. Homology modeling of MtChit5suggests a special fatty acid chain binding cleft in MtChit5. In conclusion, theobtained data provide clues that MtChit5is the searched lipodisaccharide-forming NFhydrolase in the rhizosphere of M. truncatula. This opens the perspective to testwhether the infection process of S. meliloti depends on NF concentrations andwhether MtChit5regulates NF levels in the rhizosphere. Furthermore, this thesisprovides evidence that class V chitinases of plants diverged during evolution andacquired distinct substrate specificities.