Substrate specificity and kinetic studies of nodulation protein NodL of Rhizobium leguminosarum.
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.
Bloemberg
G V
GV
Institute of Molecular Plant Sciences, Leiden University, The Netherlands.
Lagas
R M
RM
van Leeuwen
S
S
Van der Marel
G A
GA
Van Boom
J H
JH
Lugtenberg
B J
BJ
Spaink
H P
HP
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
D001426:Bacterial Proteins; D002686:Chitin; D000123:Acetyltransferases