Dataset Information

NMR structure of the enzyme GatB of the galactitol-specific phosphoenolpyruvate-dependent phosphotransferase system and its interaction with GatA.

ABSTRACT: The phosphoenolpyruvate-dependent carbohydrate transport system (PTS) couples uptake with phosphorylation of a variety of carbohydrates in prokaryotes. In this multienzyme complex, the enzyme II (EII), a carbohydrate-specific permease, is constituted of two cytoplasmic domains, IIA and IIB, and a transmembrane channel IIC domain. Among the five families of EIIs identified in Escherichia coli, the galactitol-specific transporter (II(gat)) belongs to the glucitol family and is structurally the least well-characterized. Here, we used nuclear magnetic resonance (NMR) spectroscopy to solve the three-dimensional structure of the IIB subunit (GatB). GatB consists of a central four-stranded parallel beta-sheet flanked by alpha-helices on both sides; the active site cysteine of GatB is located at the beginning of an unstructured loop between beta1 and alpha1 that folds into a P-loop-like structure. This structural arrangement shows similarities with other IIB subunits but also with mammalian low molecular weight protein tyrosine phosphatases (LMW PTPase) and arsenate reductase (ArsC). An NMR titration was performed to identify the GatA-interacting residues.

SUBMITTER: Volpon L

PROVIDER: S-EPMC2242383 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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NMR structure of the enzyme GatB of the galactitol-specific phosphoenolpyruvate-dependent phosphotransferase system and its interaction with GatA.

Volpon Laurent L Young Christopher R CR Matte Allan A Gehring Kalle K

Protein science : a publication of the Protein Society 20060908 10

The phosphoenolpyruvate-dependent carbohydrate transport system (PTS) couples uptake with phosphorylation of a variety of carbohydrates in prokaryotes. In this multienzyme complex, the enzyme II (EII), a carbohydrate-specific permease, is constituted of two cytoplasmic domains, IIA and IIB, and a transmembrane channel IIC domain. Among the five families of EIIs identified in Escherichia coli, the galactitol-specific transporter (II(gat)) belongs to the glucitol family and is structurally the lea ...[more]

PMID: 16963640

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Project description:In Vibrio cholerae, the genes required for chitin utilization and natural competence are governed by the chitin-responsive two-component system (TCS) sensor kinase ChiS. In the classical TCS paradigm, a sensor kinase specifically phosphorylates a cognate response regulator to activate gene expression. However, our previous genetic study suggested that ChiS stimulates the non-TCS transcriptional regulator TfoS by using mechanisms distinct from classical phosphorylation reactions (S. Yamamoto, J. Mitobe, T. Ishikawa, S. N. Wai, M. Ohnishi, H. Watanabe, and H. Izumiya, Mol Microbiol 91:326-347, 2014, https://doi.org/10.1111/mmi.12462). TfoS specifically activates the transcription of tfoR, encoding a small regulatory RNA essential for competence gene expression. Whether ChiS and TfoS interact directly remains unknown. To determine if other factors mediate the communication between ChiS and TfoS, we isolated transposon mutants that turned off tfoR::lacZ expression but possessed intact chiS and tfoS genes. We demonstrated an unexpected association of chitin-induced signaling pathways with the glucose-specific enzyme IIA (EIIAglc) of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses revealed that dephosphorylated EIIAglc inactivated natural competence and tfoR transcription. Chitin-induced expression of the chb operon, which is required for chitin transport and catabolism, was also repressed by dephosphorylated EIIAglc Furthermore, the regulation of tfoR and chb expression by EIIAglc was dependent on ChiS and intracellular levels of ChiS were not affected by disruption of the gene encoding EIIAglc These results define a previously unknown connection between the PTS and chitin signaling pathways in V. cholerae and suggest a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.IMPORTANCE The EIIAglc protein of the PTS coordinates a wide variety of physiological functions with carbon availability. In this report, we describe an unexpected association of chitin-activated signaling pathways in V. cholerae with EIIAglc The signaling pathways are governed by the chitin-responsive TCS sensor kinase ChiS and lead to the induction of chitin utilization and natural competence. We show that dephosphorylated EIIAglc inhibits both signaling pathways in a ChiS-dependent manner. This inhibition is different from classical catabolite repression that is caused by lowered levels of cyclic AMP. This work represents a newly identified connection between the PTS and chitin signaling pathways in V. cholerae and suggests a strategy whereby this bacterium can physiologically adapt to the existing nutrient status.

Project description:The phosphoenolpyruvate-dependent phosphotransferase system (PTS) is the main carbohydrate uptake system in Bacillus subtilis A typical PTS consists of two general proteins, enzyme I (EI) and a histidine-containing protein (HPr), as well as a specific carbohydrate transporter (or enzyme II [EII]), all of which transfer the phosphoryl group from phosphoenolpyruvate to the transported carbohydrate. The specific PTS transporters are formed by multidomain proteins or single-domain subunits. These domains are domain C (EIIC), the transmembrane channel for the carbohydrate transport; domain B (EIIB), the membrane-bound domain responsible for phosphorylation of the carbohydrate; and domain A (EIIA), the mediator between HPr(H15?P) and EIIB. There are 16 PTS transporters in B. subtilis, 6 of which, i.e., NagP, MalP, MurP, TreP, SacP, and SacX, contain no EIIA domain. Deletion of the single-EIIA-containing transporters showed that there is cross talk between the noncognate EIIA and EIIB domains in PTS. By deletion of all EIIA-containing proteins, strain KM455 (?EIIA) was constructed, and the EIIA-containing proteins were individually introduced into the strain. In this way, the PTS transporters of the glucose family, namely, PtsG, GamP, and PtsA (also known as YpqE), enabled growth with maltose, N-acetylglucosamine, sucrose, or trehalose as the sole carbon source. Construction of TkmA-EIIA fusion proteins confirmed the probable interaction between the EIIAs of the glucose family of PTS transporters and the EIIA-deficient PTS transporters. Likewise, we have shown that SacX is mainly phosphorylated by PtsA and GamP. PtsG and GmuA were also able to phosphorylate SacX, albeit less well than GamP and PtsA.IMPORTANCE The phosphoenolpyruvate-dependent phosphotransferase system (PTS) not only is a carbohydrate uptake system in B. subtilis but also plays an important role in sensing the nutrient fluctuation in the medium. This sensing system enables the cells to respond to these fluctuations properly. The PTS transporters have a pivotal role in this sensing system since they are carbohydrate specific. In this study, we tried to understand the interactions among these transporters which revealed the cross talk among PTSs. Three PTS proteins, namely, PtsG (the specific transporter of glucose), GamP (the specific transporter of glucosamine), and PtsA (a cytoplasmic single-domain EIIA protein) were shown to play the major role in the interaction among the PTSs.

Dataset Information

NMR structure of the enzyme GatB of the galactitol-specific phosphoenolpyruvate-dependent phosphotransferase system and its interaction with GatA.

Publications

NMR structure of the enzyme GatB of the galactitol-specific phosphoenolpyruvate-dependent phosphotransferase system and its interaction with GatA.

Similar Datasets

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