title: 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.
authors.affiliation: State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, East Campus, Guangzhou 510006, People's Republic of China.
authors.forename: Lan-Yue
authors.initials: LY
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, East Campus, Guangzhou 510006, People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Cai
authors.affiliation: State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, East Campus, Guangzhou 510006, People's Republic of China.
authors.forename: Ru-Jie
authors.initials: RJ
authors.lastname: Li
authors.affiliation: State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, East Campus, Guangzhou 510006, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, East Campus, Guangzhou 510006, People's Republic of China.
authors.forename: Christian
authors.initials: C
authors.lastname: Wagner
authors.affiliation: Chinese University of Hong Kong, Shatin, Hong Kong, People's Republic of China.
authors.forename: Kam-Bo
authors.initials: KB
authors.lastname: Wong
authors.affiliation: State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, East Campus, Guangzhou 510006, People's Republic of China Shenzhen Research and Development Center of State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Baoan, Shenzhen, People's Republic of China xiezping@mail.sysu.edu.cn.
authors.forename: Zhi-Ping
authors.initials: ZP
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, East Campus, Guangzhou 510006, People's Republic of China Shenzhen Research and Development Center of State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Baoan, Shenzhen, People's Republic of China cst@mail.sysu.edu.cn.
authors.forename: Christian
authors.initials: C
authors.lastname: Staehelin
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry; D002688:Chitinases / Q000235:genetics* / Q000378:metabolism*; D006868:Hydrolysis; D000070116:Lotus / Q000201:enzymology* / Q000235:genetics; D046913:Medicago truncatula / Q000201:enzymology* / Q000235:genetics / Q000382:microbiology; D009586:Nitrogen Fixation; D009844:Oligosaccharides / Q000737:chemistry*; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D016962:Sinorhizobium meliloti; D013379:Substrate Specificity; D013559:Symbiosis
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D010940:Plant Proteins; D002688:Chitinases
keywords: Medicago truncatula; Nod-factor hydrolase; Nod-factors (lipo-chitooligosaccharides); chitinase; neofunctionalization