Purification and characterization of a novel chitinase-lysozyme, of another chitinase, both hydrolysing Rhizobium meliloti Nod factors, and of a pathogenesis-related protein from Medicago sativa roots.
The symbiosis between Rhizobium meliloti and Medicago sativa (Leguminosae) involves the interaction of lipochito-oligosaccharides (Nod factors) excreted by bacteria with specific proteins of the host plant. The cleavage of Nod factors can be used as an enzymic assay to identify novel hydrolytic enzymes. Here a soluble extract of 3-day-old roots was fractionated by anion exchange, affinity chromatography, gel filtration and native electrophoresis. Two acidic chitinases (pI 4.6-5.4), CHIT24 and CHIT36, designated in accordance with their molecular mass in kDa, were separated. CHIT24 cleaves all tested Nod factors to produce lipotrisaccharides with the preference NodRm-V(S)>NodRm-IV >NodRm-IV(S)>=NodRm-IV(Ac,S); it also hydrolyses colloidal 3H-chitin and has lysozyme activity. The kinetics of Nod factor degradation by CHIT24 depends on substrate structural parameters, namely the length of the oligosaccharide chain and sulphation (S) at the reducing end, but not much on acetylation (Ac) at the non-reducing end. The 25-residue N-terminal sequence of CHIT24 has no similarity with known chitinases or lysozymes, indicating that it is a novel type of hydrolase. CHIT36 also hydrolyses NodRm-V(S) into NodRm-III, but it is inactive towards NodRm-IV(S) and NodRm-IV(Ac,S) formed by R. meliloti. Finally, a 17 kDa protein, P17, was co-purified with CHIT24. It neither degrades Nod factors nor exhibits lysozyme activity and shows complete identity, at the 15-residue N-terminal sequence, with a class 10 pathogenesis-related protein, PR-10.
Minic
Z
Z
Institut des Sciences Végétales (CNRS-UPR 40), F-91198 Gif-sur-Yvette cedex, France. minic@isv.cnrs.gif.fr
Brown
S
S
De Kouchkovsky
Y
Y
Schultze
M
M
Staehelin
C
C
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D002688:Chitinases / Q000737:chemistry*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000378:metabolism; D000455:Medicago sativa / Q000201:enzymology*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D017421:Sequence Analysis; D016962:Sinorhizobium meliloti / Q000737:chemistry; D013379:Substrate Specificity; D013559:Symbiosis / Q000502:physiology
D008070:Lipopolysaccharides; D009844:Oligosaccharides; D010940:Plant Proteins; C053376:pathogenesis-related proteins, plant; D002688:Chitinases; C113067:chitinase CHIT24