Mutations Lowering the Phosphatase Activity of HPr Kinase/phosphatase Switch off Carbon Metabolism

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 2001 Aug 3

PMID 11483496

Citations 27

Authors

V Monedero

S Poncet

I Mijakovic

S Fieulaine

V Dossonnet

I Martin-Verstraete

S Nessler

J Deutscher

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Deciphering host dynamics using dual activity-based protein profiling of ATP-interacting proteins.

Ahator S, Hegstad K, Lentz C, Johannessen M mSystems. 2024; 9(5):e0017924.

PMID: 38656122 PMC: 11097646. DOI: 10.1128/msystems.00179-24.

Identification of HPr kinase/phosphorylase inhibitors: novel antimicrobials against resistant Enterococcus faecalis.

Kumar S, Bhadane R, Shandilya S, Salo-Ahen O, Kapila S J Comput Aided Mol Des. 2022; 36(7):507-520.

PMID: 35809194 PMC: 9399212. DOI: 10.1007/s10822-022-00461-6.

Deciphering the Regulation of the Mannitol Operon Paves the Way for Efficient Production of Mannitol in Lactococcus lactis.

Xiao H, Bang-Berthelsen C, Jensen P, Solem C Appl Environ Microbiol. 2021; 87(16):e0077921.

PMID: 34105983 PMC: 8315166. DOI: 10.1128/AEM.00779-21.

Enterococcus faecalis Maltodextrin Gene Regulation by Combined Action of Maltose Gene Regulator MalR and Pleiotropic Regulator CcpA.

Grand M, Riboulet-Bisson E, Deutscher J, Hartke A, Sauvageot N Appl Environ Microbiol. 2020; 86(18).

PMID: 32680872 PMC: 7480380. DOI: 10.1128/AEM.01147-20.

Potential Regulatory Role of Competitive Encounter Complexes in Paralogous Phosphotransferase Systems.

Strickland M, Kale S, Strub M, Schwieters C, Liu J, Peterkofsky A J Mol Biol. 2019; 431(12):2331-2342.

PMID: 31071328 PMC: 6554058. DOI: 10.1016/j.jmb.2019.04.040.

References

Vagner V, Dervyn E, Ehrlich S . A vector for systematic gene inactivation in Bacillus subtilis. Microbiology (Reading). 1998; 144 ( Pt 11):3097-3104. DOI: 10.1099/00221287-144-11-3097. View

Ikeda T, LaPorte D . Isocitrate dehydrogenase kinase/phosphatase: aceK alleles that express kinase but not phosphatase activity. J Bacteriol. 1991; 173(5):1801-6. PMC: 207332. DOI: 10.1128/jb.173.5.1801-1806.1991. View

Reizer J, Hoischen C, Titgemeyer F, Rivolta C, Rabus R, Stulke J . A novel protein kinase that controls carbon catabolite repression in bacteria. Mol Microbiol. 1998; 27(6):1157-69. DOI: 10.1046/j.1365-2958.1998.00747.x. View

Hasemann C, Istvan E, Uyeda K, Deisenhofer J . The crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies. Structure. 1996; 4(9):1017-29. DOI: 10.1016/s0969-2126(96)00109-8. View

Galinier A, Longin R, Deutscher J, Danchin A, Glaser P, Martin-Verstraete I . Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis. J Bacteriol. 1999; 181(22):6889-97. PMC: 94162. DOI: 10.1128/JB.181.22.6889-6897.1999. View

Heine-Suner D, Lange A, Rodriguez de Cordoba S . Sequence and structure of the human 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase heart isoform gene (PFKFB2). Eur J Biochem. 1998; 254(1):103-10. DOI: 10.1046/j.1432-1327.1998.2540103.x. View

Huynh P, Jankovic I, Schnell N, Bruckner R . Characterization of an HPr kinase mutant of Staphylococcus xylosus. J Bacteriol. 2000; 182(7):1895-902. PMC: 101872. DOI: 10.1128/JB.182.7.1895-1902.2000. View

Viana R, Monedero V, Dossonnet V, Vadeboncoeur C, Perez-Martinez G, Deutscher J . Enzyme I and HPr from Lactobacillus casei: their role in sugar transport, carbon catabolite repression and inducer exclusion. Mol Microbiol. 2000; 36(3):570-84. DOI: 10.1046/j.1365-2958.2000.01862.x. View

Eisermann R, Deutscher J, Hengstenberg W . Site-directed mutagenesis with the ptsH gene of Bacillus subtilis. Isolation and characterization of heat-stable proteins altered at the ATP-dependent regulatory phosphorylation site. J Biol Chem. 1988; 263(32):17050-4. View

10.

Henkin T, Grundy F, Nicholson W, Chambliss G . Catabolite repression of alpha-amylase gene expression in Bacillus subtilis involves a trans-acting gene product homologous to the Escherichia coli lacl and galR repressors. Mol Microbiol. 1991; 5(3):575-84. DOI: 10.1111/j.1365-2958.1991.tb00728.x. View

11.

LaPorte D, Koshland Jr D . A protein with kinase and phosphatase activities involved in regulation of tricarboxylic acid cycle. Nature. 1982; 300(5891):458-60. DOI: 10.1038/300458a0. View

12.

Stulke J, Hillen W . Regulation of carbon catabolism in Bacillus species. Annu Rev Microbiol. 2000; 54:849-80. DOI: 10.1146/annurev.micro.54.1.849. View

13.

Deutscher J, Saier Jr M . ATP-dependent protein kinase-catalyzed phosphorylation of a seryl residue in HPr, a phosphate carrier protein of the phosphotransferase system in Streptococcus pyogenes. Proc Natl Acad Sci U S A. 1983; 80(22):6790-4. PMC: 390071. DOI: 10.1073/pnas.80.22.6790. View

14.

Ye J, Saier Jr M . Regulation of sugar uptake via the phosphoenolpyruvate-dependent phosphotransferase systems in Bacillus subtilis and Lactococcus lactis is mediated by ATP-dependent phosphorylation of seryl residue 46 in HPr. J Bacteriol. 1996; 178(12):3557-63. PMC: 178126. DOI: 10.1128/jb.178.12.3557-3563.1996. View

15.

Thompson J, Torchia D . Use of 31P nuclear magnetic resonance spectroscopy and 14C fluorography in studies of glycolysis and regulation of pyruvate kinase in Streptococcus lactis. J Bacteriol. 1984; 158(3):791-800. PMC: 215511. DOI: 10.1128/jb.158.3.791-800.1984. View

16.

Draborg H, Villadsen D, Nielsen T . Cloning, characterization and expression of a bifunctional fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase from potato. Plant Mol Biol. 1999; 39(4):709-20. DOI: 10.1023/a:1006102412693. View

17.

Garcia E, Rhee S . Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII uridylyltransferase and uridylyl-removing enzyme. J Biol Chem. 1983; 258(4):2246-53. View

18.

Jaggi R, van Heeswijk W, Westerhoff H, Ollis D, Vasudevan S . The two opposing activities of adenylyl transferase reside in distinct homologous domains, with intramolecular signal transduction. EMBO J. 1997; 16(18):5562-71. PMC: 1170188. DOI: 10.1093/emboj/16.18.5562. View

19.

Stulke J, Martin-Verstraete I, Zagorec M, Rose M, Klier A, Rapoport G . Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT. Mol Microbiol. 1997; 25(1):65-78. DOI: 10.1046/j.1365-2958.1997.4351797.x. View

20.

Galinier A, Deutscher J, Martin-Verstraete I . Phosphorylation of either crh or HPr mediates binding of CcpA to the bacillus subtilis xyn cre and catabolite repression of the xyn operon. J Mol Biol. 1999; 286(2):307-14. DOI: 10.1006/jmbi.1998.2492. View