Abstract

The phosphoenolpyruvate: glycose phosphotransferase system (PTS) catalyzes the uptake and concomitant phosphorylation of D-glucose (GIc) and many other sugars by Escherichia coli, Salmonella, Vibrios, and numerous other pathogens, especially obligate anaerobes. Since GIc is catabolized as rapidly as it is translocated, the PTS determines the rate of cell growth in GIc-Limiting media Despite an extensive literature, little is known about the molecular details of each step, and virtually nothing on how the PTS is so stringently regulated. We have recently developed a rapid quench procedure that will permit detailed biochemical studies of each step, and may ultimately lead to an understanding of how the PTS is regulated. The method has already shown that the first step, autophosphorylation of EI by PEP, is very complex because of the slow El monomer/dimer (NM) transition. EI may be a major regulator of GIc uptake via the MAD transition, and/or a recently discovered, ATP-dependent El kinase that may link PTS to the electron transport chain. Biochemical studies on both systems will be continued. One goal will be to determine the ratio of M/D during GIc uptake by membrane vesicles using fluorescence energy transfer and EI mutants. A second function of the PTS is that it mediates the """"""""glucose effect"""""""". In this signal transduction system, external GIc repressed expression of certain catabolic operons, and the protein IIP' is the effector, 111' interacts with at least 10 other proteins, including non-PTS perineases. The structures of HI' and important mutants have been well established, as has the complex between HI' and one of its targets, glycerol kinase (GK). We have now constructed 15 crr (HIC*) mutants, and these will be used to further probe interactions between this key regulatory molecule and P-BPr, and GK. For example, application of the rapid quench method to a HI' mutant has suggested a possible mechanism for the P-transfer reaction between BPr -- RIC. These studies should ultimately provide a molecular explanation for sugar uptake by bacterial pathogens.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038759-14
Application #
6179537
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Shapiro, Bert I
Project Start
1987-09-01
Project End
2001-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
14
Fiscal Year
2000
Total Cost
$580,211
Indirect Cost

  Institution

Name
Johns Hopkins University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218

  Publications

Xu, Jianhua; Chen, Jiejin; Toptygin, Dmitri et al. (2009) Femtosecond fluorescence spectra of tryptophan in human gamma-crystallin mutants: site-dependent ultrafast quenching. J Am Chem Soc 131:16751-7
Xu, Jianhua; Toptygin, Dmitri; Graver, Karen J et al. (2006) Ultrafast fluorescence dynamics of tryptophan in the proteins monellin and IIAGlc. J Am Chem Soc 128:1214-21
Patel, Himatkumar V; Vyas, Kavita A; Mattoo, Roshan L et al. (2006) Properties of the C-terminal domain of enzyme I of the Escherichia coli phosphotransferase system. J Biol Chem 281:17579-87
Patel, Himatkumar V; Vyas, Kavita A; Savtchenko, Regina et al. (2006) The monomer/dimer transition of enzyme I of the Escherichia coli phosphotransferase system. J Biol Chem 281:17570-8
Meadow, Norman D; Mattoo, Roshan L; Savtchenko, Regina S et al. (2005) Transient state kinetics of Enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system of Escherichia coli: equilibrium and second-order rate constants for the phosphotransfer reactions with phosphoenolpyruvate and HPr. Biochemistry 44:12790-6
Meibom, Karin L; Li, Xibing B; Nielsen, Alex T et al. (2004) The Vibrio cholerae chitin utilization program. Proc Natl Acad Sci U S A 101:2524-9
Patel, Himatkumar V; Vyas, Kavita A; Li, Xibing et al. (2004) Subcellular distribution of enzyme I of the Escherichia coli phosphoenolpyruvate:glycose phosphotransferase system depends on growth conditions. Proc Natl Acad Sci U S A 101:17486-91
Holtman, C K; Pawlyk, A C; Meadow, N D et al. (2001) Reverse genetics of Escherichia coli glycerol kinase allosteric regulation and glucose control of glycerol utilization in vivo. J Bacteriol 183:3336-44
Holtman, C K; Pawlyk, A C; Meadow, N et al. (2001) IIA(Glc) allosteric control of Escherichia coli glycerol kinase: binding site cooperative transitions and cation-promoted association by Zinc(II). Biochemistry 40:14302-8
Keyhani, N O; Wang, L X; Lee, Y C et al. (2000) The chitin disaccharide, N,N'-diacetylchitobiose, is catabolized by Escherichia coli and is transported/phosphorylated by the phosphoenolpyruvate:glycose phosphotransferase system. J Biol Chem 275:33084-90

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