Mijakovic Ivan, Poncet Sandrine, Galinier Anne, Monedero Vicente, Fieulaine Sonia, Janin Joël, Nessler Sylvie, Marquez José Antonio, Scheffzek Klaus, Hasenbein Sonja, Hengstenberg Wolfgang, Deutscher Josef
Laboratoire de Génétique des Microorganismes, Centre National de la Recherche Scientifique, Unité de Recherche Associée 1925, F-78850 Thiverval-Grignon, France.
Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13442-7. doi: 10.1073/pnas.212410399. Epub 2002 Oct 1.
In most Gram-positive bacteria, serine-46-phosphorylated HPr (P-Ser-HPr) controls the expression of numerous catabolic genes ( approximately 10% of their genome) by acting as catabolite corepressor. HPr kinase/phosphorylase (HprK/P), the bifunctional sensor enzyme for catabolite repression, phosphorylates HPr, a phosphocarrier protein of the sugar-transporting phosphoenolpyruvate/glycose phosphotransferase system, in the presence of ATP and fructose-1,6-bisphosphate but dephosphorylates P-Ser-HPr when phosphate prevails over ATP and fructose-1,6-bisphosphate. We demonstrate here that P-Ser-HPr dephosphorylation leads to the formation of HPr and pyrophosphate. HprK/P, which binds phosphate at the same site as the beta phosphate of ATP, probably uses the inorganic phosphate to carry out a nucleophilic attack on the phosphoryl bond in P-Ser-HPr. HprK/P is the first enzyme known to catalyze P-protein dephosphorylation via this phospho-phosphorolysis mechanism. This reaction is reversible, and at elevated pyrophosphate concentrations, HprK/P can use pyrophosphate to phosphorylate HPr. Growth of Bacillus subtilis on glucose increased intracellular pyrophosphate to concentrations ( approximately 6 mM), which in in vitro tests allowed efficient pyrophosphate-dependent HPr phosphorylation. To effectively dephosphorylate P-Ser-HPr when glucose is exhausted, the pyrophosphate concentration in the cells is lowered to 1 mM. In B. subtilis, this might be achieved by YvoE. This protein exhibits pyrophosphatase activity, and its gene is organized in an operon with hprK.
在大多数革兰氏阳性菌中，丝氨酸46位磷酸化的HPr（P-Ser-HPr）作为分解代谢物共阻遏物，控制着众多分解代谢基因（约占其基因组的10%）的表达。HPr激酶/磷酸酶（HprK/P）是分解代谢物阻遏的双功能传感酶，在ATP和果糖-1,6-二磷酸存在时，将糖转运磷酸烯醇丙酮酸/葡萄糖磷酸转移酶系统的磷酸载体蛋白HPr磷酸化，但当磷酸盐含量超过ATP和果糖-1,6-二磷酸时，会使P-Ser-HPr去磷酸化。我们在此证明，P-Ser-HPr去磷酸化会导致HPr和焦磷酸的形成。HprK/P在与ATP的β磷酸相同的位点结合磷酸盐，可能利用无机磷酸盐对P-Ser-HPr中的磷酰键进行亲核攻击。HprK/P是已知的第一种通过这种磷-磷解机制催化磷蛋白去磷酸化的酶。该反应是可逆的，在焦磷酸盐浓度升高时，HprK/P可以利用焦磷酸盐使HPr磷酸化。枯草芽孢杆菌在葡萄糖上生长会使细胞内焦磷酸盐浓度增加到（约6 mM），在体外试验中，这允许焦磷酸盐依赖性的HPr高效磷酸化。当葡萄糖耗尽时，为了有效使P-Ser-HPr去磷酸化，细胞内的焦磷酸盐浓度会降低到1 mM。在枯草芽孢杆菌中，这可能是由YvoE实现的。该蛋白具有焦磷酸酶活性，其基因与hprK位于一个操纵子中。
