RESUMO
Bacteria sense continuous changes in their environment and adapt metabolically to effectively compete with other organisms for limiting nutrients. One system which plays an important part in this adaptation response is the phosphoenol-pyruvate:sugar phosphotransferase system (PTS). Many proteins interact with and are regulated by PTS components in bacteria. Here we review the interaction with and allosteric regulation of Escherichia coli glycogen phosphorylase (GP) activity by the histidine phosphocarrier protein HPr, which acts as part of a phosphoryl shuttle between enzyme I and sugar-specific proteins of the PTS. HPr mediates crosstalk between PTS sugar uptake and glycogen breakdown. The evolution of the allosteric regulation of E. coli GP by HPr is compared to that of other phosphorylases.
Assuntos
Proteínas de Bactérias , Escherichia coli/genética , Escherichia coli/metabolismo , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Fosforilases/genética , Fosforilases/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Fosforilases/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The unphosphorylated form of enzyme IIAglc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system inhibits transport catalyzed by lactose permease. We (Seok et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 13515-13519) previously characterized the area on the cytoplasmic face of lactose permease that interacts with enzyme IIAglc, using radioactive enzyme IIAglc. Subsequent studies (Sondej et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530) suggested consensus binding sequences on proteins that interact with enzyme IIAglc. The present study characterizes a region on the surface of enzyme IIAglc that interfaces with lactose permease. Acetylation of lysine residues by sulfosuccinimidyl acetate treatment of enzyme IIAglc, but not lactose permease, reduced the degree of interaction between the two proteins. To localize the lysine residue(s) on enzyme IIAglc that is(are) involved in the regulatory interaction, selected lysine residues were mutagenized. Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr. Except for Lys69, all the modified proteins were as effective as the wild-type enzyme IIAglc in a test for binding to lactose permease. The Lys69 mutant was also defective in phosphoryl transfer to glucose permease. To derive further information concerning the contact surface, additional selected residues in the vicinity of Lys69 were mutagenized and tested for binding to lactose permease. On the basis of these studies, a model for the region of the surface of enzyme IIAglc that interacts with lactose permease is proposed.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Monossacarídeos , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Simportadores , Acetatos/farmacologia , Acetilação , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/fisiologia , Lisina/genética , Lisina/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/antagonistas & inibidores , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Succinimidas/farmacologiaRESUMO
Products of the pts operon of Escherichia coli have multiple physiological roles such as sugar transport, and the operon is controlled by two promoters, P0 and P1. Expression of the pts P0 promoter that is increased during growth in the presence of glucose is also activated by cAMP receptor protein.cAMP. Based on the existence of a sequence that has a high similarity with the known Mlc binding site in the promoter, the effects of the Mlc protein on the pts P0 promoter expression were studied. In vivo transcription assays using wild type and mlc-negative E. coli strains grown in the presence and absence of glucose indicate that Mlc negatively regulates expression of the P0 promoter, and Mlc-dependent repression is relieved by glucose in the growth medium. In vitro transcription assay using purified recombinant Mlc showed that Mlc repressed transcription from the P0 but did not affect the activity of the P1. DNase I footprinting experiments revealed that a Mlc binding site was located around +1 to +25 of the promoter and that Mlc inhibited the binding of RNA polymerase to the P0 promoter. Cells overexpressing Mlc showed a very slow fermentation rate compared with the wild type when grown in the presence of various phosphoenolpyruvate-carbohydrate phosphotransferase system sugars but few differences in the presence of non-phosphoenolpyruvate-carbohydrate phosphotransferase system sugars except maltose. These results suggest that the pts operon is one of major targets for the negative regulation by Mlc, and thus Mlc regulates the utilization of various sugars as well as glucose in E. coli. The possibility that the inducer of Mlc may not be sugar or its derivative but an unknown factor is proposed to explain the Mlc induction mechanism by various sugars.
Assuntos
Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/biossíntese , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Escherichia coli , Dados de Sequência Molecular , Regiões Promotoras GenéticasRESUMO
Enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system undergoes a slow monomer-dimer transition. In vitro autophosphorylation of Enzyme I by PEP was studied at limiting concentrations of the protein. Addition to incubation mixtures containing wild-type Enzyme I of inactive or low-activity mutant forms of Enzyme I resulted in stimulation of autophosphorylation activity. The kinetics of the activation fit well to a model in which the active form of Enzyme I is the dimer. These experiments provide support for the argument that only the dimeric form of Enzyme I can be autophosphorylated.