Monedero V, Poncet S, Mijakovic I, Fieulaine S, Dossonnet V, Martin-Verstraete I, Nessler S, Deutscher J
Laboratoire de Génétique des Microorganismes, INRA and CNRS URA1925, Thiverval-Grignon, France.
EMBO J. 2001 Aug 1;20(15):3928-37. doi: 10.1093/emboj/20.15.3928.
The oligomeric bifunctional HPr kinase/P-Ser-HPr phosphatase (HprK/P) regulates many metabolic functions in Gram-positive bacteria by phosphorylating the phosphocarrier protein HPr at Ser46. We isolated Lactobacillus casei hprK alleles encoding mutant HprK/Ps exhibiting strongly reduced phosphatase, but almost normal kinase activity. Two mutations affected the Walker motif A of HprK/P and four a conserved C-terminal region in contact with the ATP-binding site of an adjacent subunit in the hexamer. Kinase and phosphatase activity appeared to be closely associated and linked to the Walker motif A, but dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr) is not simply a reversal of the kinase reaction. When the hprKV267F allele was expressed in Bacillus subtilis, the strongly reduced phosphatase activity of the mutant enzyme led to increased amounts of P-Ser-HPr. The hprKV267F mutant was unable to grow on carbohydrates transported by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and on most non-PTS carbohydrates. Disrupting ccpA relieved the growth defect only on non-PTS sugars, whereas replacing Ser46 in HPr with alanine also restored growth on PTS substrates.
寡聚双功能HPr激酶/磷酸化丝氨酸-HPr磷酸酶（HprK/P）通过在丝氨酸46位点磷酸化磷酸载体蛋白HPr来调节革兰氏阳性菌中的多种代谢功能。我们分离出了干酪乳杆菌hprK等位基因，其编码的突变型HprK/P表现出磷酸酶活性大幅降低，但激酶活性几乎正常。两个突变影响了HprK/P的沃克基序A，四个突变影响了与六聚体中相邻亚基的ATP结合位点接触的保守C末端区域。激酶和磷酸酶活性似乎紧密相关并与沃克基序A相连，但丝氨酰磷酸化HPr（P-Ser-HPr）的去磷酸化并非简单地是激酶反应的逆转。当hprKV267F等位基因在枯草芽孢杆菌中表达时，突变酶大幅降低的磷酸酶活性导致P-Ser-HPr的量增加。hprKV267F突变体无法在磷酸烯醇丙酮酸：葡萄糖磷酸转移酶系统（PTS）转运的碳水化合物以及大多数非PTS碳水化合物上生长。破坏ccpA仅能缓解在非PTS糖上的生长缺陷，而将HPr中的丝氨酸46替换为丙氨酸也能恢复在PTS底物上的生长。
