doi:10.1016/j.pep.2007.04.012
Copyright © 2007 Elsevier Inc. All rights reserved.
Purification and characterization of the bifunctional uridylyltransferase and the signal transducing proteins GlnB and GlnK from Herbaspirillum seropedicae
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Ana C. Bonattoa, Gustavo H. Coutoa, Emanuel M. Souzaa, Luiza M. Araújoa, Fabio O. Pedrosaa, Lilian Noindorfa and Elaine M. Benelli
, a, 
aDepartment of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP19046 Curitiba, PR, Brazil
Received 14 March 2007;
revised 9 April 2007.
Available online 25 April 2007.
Abstract
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.
Keywords: GlnD; GlnB; GlnK; Uridylylation; Herbaspirillum seropedicae
Fig. 1. Expression of H. seropedicae GlnB (a), GlnK (b) and GlnD (c) proteins in E. coli RB9065λDE3. (a) Crude extract of cells carrying pET29a+ (lane 1) or pEMB101.8 (lane 2). (b) Crude extract of cells carrying pET29a+ (lane 1) or pEMB200 (lane 2). (c) Crude extract of cells carrying pET29a+ (lane 1) or pGH2 (lane 2). Expression was induced with 0.5 mM IPTG for 3 h (GlnB and GlnK) or 4 h (GlnD). Arrows indicate overexpressed proteins. MW indicates molecular weight markers in kDa (Fermentas). Proteins were stained with Coomassie blue R250.
Fig. 2. Expression of H. seropedicae GlnD protein in E. coli RB9065λDE3. Soluble (lane 1) and insoluble (lane 2) fractions after induction at 30 °C; soluble (lane 3) and insoluble (lane 4) fractions after induction at 37 °C. GlnD expression was induced from plasmid pGH2 with 0.5 mM IPTG. The overexpressed proteins are indicated by arrows. MW indicates molecular weight markers in kDa (GE Healthcare). Proteins were stained with Coomassie blue R250.
Fig. 3. Herbaspirillum seropedicae GlnB, GlnK and GlnD purified proteins. (a) GlnB protein (lane 1) and GlnK protein (lane 2). (b) GlnD protein. The purified proteins are indicated by arrows. MW indicates molecular weight markers in kDa (GE Healthcare). Proteins were silver stained.
Fig. 4. Uridylylation of H. seropedicae GlnB and GlnK proteins. Reactions were carried out as described in Materials and methods and loaded on non-denaturing polyacrylamide gels. Lane 1, uridylylation reaction containing ATP (200 μM), 2-oxoglutarate (5 mM), UTP (1 mM), purified proteins GlnB (2 μM) or GlnK (2 μM) and GlnD (100 nM) stopped at time 0; lane 2, complete reaction (as in 1) but with 50 nM GlnD and incubated for 10 min; lane 3, complete reaction incubated for 10 min; lane 4, reaction in the absence of UTP; lane 5, reaction in the absence of GlnD; lane 6, reaction in the absence of GlnB or GlnK. lane 7, reaction in the absence of ATP; lane 8, reaction in the absence of 2-oxoglutarate.
Fig. 5. Inhibition of the uridylylation of H. seropedicae GlnB and GlnK proteins by glutamine. Reactions were carried out as described in Materials and methods and contained ATP (200 μM), 2-oxoglutarate (5 mM), UTP (1 mM), purified proteins GlnB (2 μM) or GlnK (2 μM) and GlnD (100 nM). (a) GlnB protein. (b) GlnK protein. Squares: no glutamine; circles: 5 mM l-glutamine.
Table 1.
Bacterial strains and plasmids used in this work
Table 2.
Purification of GlnB, GlnK and GlnD proteins of H. seropedicae from E. coli overproducing strains
Total protein was measured by the Bradford method [38]. Purity was determined by densitometric analyses of purification step samples loaded on SDS–PAGE and stained with Coomassie blue R250. Yield was calculated as the ratio of the amount of purified protein achieved and the amount present in the previous step.
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