PII-like proteins are signal transduction proteins found in species from all kingdoms. In Escherichia coli and Klebsiella pneumoniae two PII-like proteins have been identified: GlnB and GlnK. The GlnB protein integrates the signals of nitrogen, carbon and energy, and transmits this information to other proteins involved in nitrogen metabolism. In these organisms, GlnB is modified by the GlnD protein (uridylyltransferase/uridylyl-removing enzyme): under low levels of glutamine GlnB is uridylylated; conversely, high glutamine levels induces deuridylylation of GlnB. In this work, the GlnB and GlnD proteins from H. seropedicae were biochemically characterised. The H. seropedicae native GlnB and GlnD proteins were overexpressed in E. coli from the plasmids pEMB101.8 and pGH2, respectively. GlnB was purified by ion exchange followed by gel filtration chromatography, and GlnD by ion exchange followed by affinity chromatography. These proteins were used in in vitro uridylylation assays. The uridylylation state of GlnB was determined by discontinuous native gel electrophoresis stained with the fluorescent dye SyPro Red. The results show that H. seropedicae GlnD adds UMP to the GlnB trimer producing GlnB (UMP)₁, GlnB (UMP)₂ and GlnB (UMP)₃ forms, in the presence of 2-oxoglutarate (10mmol/L) and ATP (1 mmol/L). The uridylylation reaction is UTP-dependent and the observed Km for UTP was approximately 70 mmol/L.
Supported by CNPq, CAPES and Fundação Araucária/Paraná Tecnologia