The Biology of Cyclic Nucleotides in E coli

Peterkofsky, A.

Abstract

The activity of the enzyme responsible for the synthesis of cyclic AMP in Escherichia coli is regulated in a complex manner that is not completely understood. Major protein factors involved in this regulation are components of the sugar transport system known as the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Consequently, considerable attention has been focused on protein components of the PTS. Nuclear magnetic resonance spectroscopy (NMR) has been used to elucidate the solution structure of the aminoterminal domain of enzyme I (EIN) of the PTS. The data show that the structure in solution is essentially identical to that in the recently elucidated X-ray crystal structure. NMR has also been used to characterize the interaction of EIN with the histidine-containing phosphocarrier protein HPr of the PTS. The binding surface on EIN is primarily located in the alpha-domain. A model of the EIN/HPr complex was proposed in which helix 1 and the helical loop of HPr slip between the two pairs of helices constituting the alpha-domain of EIN. A functional role for the kink between helices H2 and H2' is proposed where it provides a flexible joint for the interaction to take place. Specificity studies of full-length enzyme I compared to the aminoterminal domain of enzyme I established that E. coli enzyme I will effect phosphoryl transfer specifically to HPr from E. coli. However, the aminoterminal domain of enzyme I shows a relaxed specificity and will effect phosphotransfer to HPrs from several bacterial species. These studies demonstrate that the carboxyl-terminal domain of enzyme I confers on the protein the capability to accept a phosphoryl group from phosphoenolpyruvate as well as a discriminator function that allows the intact protein to promote effective phosphoryl transfer only to E. coli HPr. Characterization of the region of the genome of Mycoplasma capricolum upstream of the portion encompassing the genes for enzymes I and IIAglc of the PTS showed that it contained a gene cluster encoding a family of genes responsible for the metabolim of pyruvate. The gene encoding enzyme IIAglc of the PTS of M. capricolum was expressed, purified and the protein characterized. It has a higher pI than previously characterized enzymes IIAglc and is unreactive against antibodies directed against the corresponding proteins from Gram-positive or Gram-negative bacteria.

Funding Agency

• Agency: National Institute of Health (NIH)
• Institute: National Heart, Lung, and Blood Institute (NHLBI)
• Type: Intramural Research (Z01)
• Project #: 1Z01HL000151-27
• Application #: 6162628
• Study Section: Special Emphasis Panel (LBG)
• Support Year: 27
• Fiscal Year: 1997

Institution

• Name: National Heart, Lung, and Blood Institute
• Country: United States

Publications

Wang, Guangshun; Peterkofsky, Alan; Keifer, Paul A et al. (2005) NMR characterization of the Escherichia coli nitrogen regulatory protein IIANtr in solution and interaction with its partner protein, NPr. Protein Sci 14:1082-90

Williams Jr, David C; Cai, Mengli; Suh, Jeong-Yong et al. (2005) Solution NMR structure of the 48-kDa IIAMannose-HPr complex of the Escherichia coli mannose phosphotransferase system. J Biol Chem 280:20775-84

Koo, Byoung-Mo; Yoon, Mi-Jeong; Lee, Chang-Ro et al. (2004) A novel fermentation/respiration switch protein regulated by enzyme IIAGlc in Escherichia coli. J Biol Chem 279:31613-21

Keifer, Paul A; Peterkofsky, Alan; Wang, Guangshun (2004) Effects of detergent alkyl chain length and chemical structure on the properties of a micelle-bound bacterial membrane targeting peptide. Anal Biochem 331:33-9

Legler, Patricia M; Cai, Mengli; Peterkofsky, Alan et al. (2004) Three-dimensional solution structure of the cytoplasmic B domain of the mannitol transporter IImannitol of the Escherichia coli phosphotransferase system. J Biol Chem 279:39115-21

Sondej, Melissa; Vazquez-Ibar, Jose Luis; Farshidi, Arta et al. (2003) Characterization of a lactose permease mutant that binds IIAGlc in the absence of ligand. Biochemistry 42:9153-9

Dimitrova, Mariana N; Peterkofsky, Alan; Ginsburg, Ann (2003) Opposing effects of phosphoenolpyruvate and pyruvate with Mg(2+) on the conformational stability and dimerization of phosphotransferase enzyme I from Escherichia coli. Protein Sci 12:2047-56

Wang, Guangshun; Keifer, Paul A; Peterkofsky, Alan (2003) Solution structure of the N-terminal amphitropic domain of Escherichia coli glucose-specific enzyme IIA in membrane-mimetic micelles. Protein Sci 12:1087-96

Li, Xia; Peterkofsky, Alan; Wang, Guangshun (2003) 1H, 15N, and 13C chemical shift assignments of the Escherichia coli nitrogen regulatory phosphocarrier IIA(Ntr). J Biomol NMR 27:401-2

Reddy, Prasad T; Prasad, C Rama; Reddy, P Hemalatha et al. (2003) Cloning and expression of the gene for a novel protein from Mycobacterium smegmatis with functional similarity to eukaryotic calmodulin. J Bacteriol 185:5263-8