title: Substrate specificity and kinetic studies of nodulation protein NodL of Rhizobium leguminosarum.
abstract: All lipo-chitin oligosaccharides identified from Rhizobium leguminosarum carry an O-acetyl moiety on C6 of the nonreducing terminal N-acetylglucosamine residue. Previously, we have shown that purified NodL protein, using acetyl-CoA as acetyl donor, in vitro acetylates N-acetylglucosamine, chitin oligosaccharides, and lipo-chitin oligosaccharides. In this paper, the enzymatic properties and substrate specificity of NodL protein were analyzed, using a spectrophotometric assay to quantify NodL transacetylating activity. NodL functions optimally under alkaline conditions. Transacetylating activity has a broad temperature optimum between 28 and 42 degrees C. NodL protein is stable for at least 15 min up to 48 degrees C. Glucosamine, chitosan oligosaccharides, terminally de-N-acetylated chitin derivatives, and cellopentaose were identified as acetyl-accepting substrates for NodL protein. Quantitative substrate specificity studies show that chitin derivatives with a free amino group on the nonreducing terminal residue are the preferred substrates of the NodL protein. Our results strongly indicate that the nonreducing terminally de-N-acetylated chitin oligosaccharides produced by the NodC and NodB enzymes are the in vivo acetyl-accepting substrates for NodL protein.
authors.affiliation: Institute of Molecular Plant Sciences, Leiden University, The Netherlands.
authors.forename: G V
authors.initials: GV
authors.lastname: Bloemberg
authors.forename: R M
authors.initials: RM
authors.lastname: Lagas
authors.forename: S
authors.initials: S
authors.lastname: van Leeuwen
authors.forename: G A
authors.initials: GA
authors.lastname: Van der Marel
authors.forename: J H
authors.initials: JH
authors.lastname: Van Boom
authors.forename: B J
authors.initials: BJ
authors.lastname: Lugtenberg
authors.forename: H P
authors.initials: HP
authors.lastname: Spaink
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002686:Chitin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation; D016961:Rhizobium leguminosarum / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002686:Chitin; D000123:Acetyltransferases