A single amino acid substitution in a chitinase of the legume Medicago truncatula is sufficient to gain Nod-factor hydrolase activity

Abstract

The symbiotic interaction between nitrogen-fixing rhizobia and legumes depends on lipo-chitooligosaccharidic Nod-factors (NFs). The NF hydrolase MtNFH1 of Medicago truncatula is a symbiotic enzyme that hydrolytically inactivates NFs with a C16 : 2 acyl chain produced by the microsymbiont Sinorhizobium meliloti 1021. MtNFH1 is related to class V chitinases (glycoside hydrolase family 18) but lacks chitinase activity. Here, we investigated the substrate specificity of MtNFH1-related proteins. MtCHIT5a and MtCHIT5b of M. truncatula as well as LjCHIT5 of Lotus japonicus showed chitinase activity, suggesting a role in plant defence. The enzymes failed to hydrolyse NFs from S. meliloti. NFs from Rhizobium leguminosarum with a C18 : 4 acyl moiety were neither hydrolysed by these chitinases nor by MtNFH1. Construction of chimeric proteins and further amino acid replacements in MtCHIT5b were performed to identify chitinase variants that gained the ability to hydrolyse NFs. A single serine-to-proline substitution was sufficient to convert MtCHIT5b into an NF-cleaving enzyme. MtNFH1 with the corresponding proline-to-serine substitution failed to hydrolyse NFs. These results are in agreement with a substrate-enzyme model that predicts NF cleavage when the C16 : 2 moiety is placed into a distinct fatty acid-binding cleft. Our findings support the view that MtNFH1 evolved from the ancestral MtCHIT5b by gene duplication and subsequent symbiosis-related neofunctionalization.

Role of the Nod Factor Hydrolase MtNFH1 in Regulating Nod Factor Levels during Rhizobial Infection and in Mature Nodules of Medicago truncatula

“…Activity of MtNFH1 depends on two loops that likely contribute to the formation of a fatty acid binding cleft for the NF substrate. Remarkably, MtCHIT5b with a single serine-toproline substitution gains the capacity to hydrolyze NFs (Tian et al, 2013;Zhang et al, 2016). MtCHIT5b and MtNFH1 are located in tandem on chromosome 4 of M. truncatula, suggesting that MtNFH1 evolved from MtCHIT5b by gene duplication.…”

“…MtCHIT5b and MtNFH1 are located in tandem on chromosome 4 of M. truncatula, suggesting that MtNFH1 evolved from MtCHIT5b by gene duplication. The evolution of a NF hydrolase from a chitinase can be considered an example of symbiosis-related neofunctionalization (Zhang et al, 2016). Transcripts of MtNFH1 are elevated during symbiosis with S. meliloti, namely, in root hairs, but also at the stage of mature nodules (Salzer et al, 2004;He et al, 2009;Breakspear et al, 2014;Roux et al, 2014;Jardinaud et al, 2016).…”

A plant chitinase controls cortical infection thread progression and nitrogen-fixing symbiosis

“…The proline residue mutated in chit5- 2 allele is located immediately after the catalytic site, and is conserved across GH18-type chitinases, indicating a crucial role for the function of this protein ( Figure 3—figure supplement 5 ). CHIT5 shares a high level of similarity to Medicago truncatula NFH1 ( Zhang et al, 2016 ), including the presence of A and B loops and the proline residue (P 294 ), previously reported as essential for Nod factor hydrolysis ( Zhang et al, 2016 ). Furthermore, we found that a version of CHIT5 mutated in the DxDxE motif (E 166 to K) was no longer able to complement the symbiotic defective phenotype, suggesting that enzymatic activity is required for CHIT5 function in planta ( Figure 3—figure supplement 6 ).…”

Chitin Synthesis and Degradation in Crustaceans: A Genomic View and Application

“…Chitinases (CHTs) comprise a group of glycoside hydrolases that cleave β-1,4-glycosidic bonds to hydrolyze chitin into chitin-oligosaccharides (GlcNAc) n and GlcNAc [ 39 ]. These enzymes have been isolated from a wide variety of sources including viruses, bacteria, fungi, nematodes, arthropods, vertebrates, and green plants, and play important roles in various physiological functions [ 30 , 38 ].…”