Purification and characterization of the bifunctional uridylyltransferase and the signal transducing proteins GlnB and GlnK from Herbaspirillum seropedicae.
GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme that has a central role in the general nitrogen regulatory system NTR. In enterobacteria, GlnD uridylylates the PII proteins GlnB and GlnK under low levels of fixed nitrogen or ammonium. Under high ammonium levels, GlnD removes UMP from these proteins (deuridylylation). The PII proteins are signal transduction elements that integrate the signals of nitrogen, carbon and energy, and transduce this information to proteins involved in nitrogen metabolism. In Herbaspirillum seropedicae, an endophytic diazotroph isolated from grasses, several genes coding for proteins involved in nitrogen metabolism have been identified and cloned, including glnB, glnK and glnD. In this work, the GlnB, GlnK and GlnD proteins of H. seropedicae were overexpressed in their native forms, purified and used to reconstitute the uridylylation system in vitro. The results show that H. seropedicae GlnD uridylylates GlnB and GlnK trimers producing the forms PII (UMP)(1), PII (UMP)(2) and PII (UMP)(3), in a reaction that requires 2-oxoglutarate and ATP, and is inhibited by glutamine. The quantification of these PII forms indicates that GlnB was more efficiently uridylylated than GlnK in the system used.
Bonatto
Ana C
AC
Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP19046 Curitiba, PR, Brazil.
Couto
Gustavo H
GH
Souza
Emanuel M
EM
Araújo
Luiza M
LM
Pedrosa
Fabio O
FO
Noindorf
Lilian
L
Benelli
Elaine M
EM
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D042803:Herbaspirillum / Q000201:enzymology / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins / Q000302:isolation & purification* / Q000378:metabolism; D015398:Signal Transduction; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D051922:PII Nitrogen Regulatory Proteins; C404143:PIID regulatory protein, Bacteria; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase