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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.37, #3)
Signal transduction and adaptive regulation through bacterial two-component systems: the Escherichia coli AtoSC paradigm by Dimitrios A. Kyriakidis; Ekaterini Tiligada (pp. 443-458).
Adaptive signal transduction within microbial cells involves a multi-faceted regulated phosphotransfer mechanism that comprises structural rearrangements of sensor histidine kinases upon ligand-binding and phosphorylation-induced conformational changes in response regulators of versatile two-component systems (TCS), arisen early in bacterial evolution. In Escherichia coli, cross-talk between the AtoS histidine kinase and the AtoC response regulator, forming the AtoSC TCS, through His → Asp phosphotransfer, activates AtoC directly to induce atoDAEB operon expression, thus modulating diverse fundamental cellular processes such as short-chain fatty acid catabolism, poly-(R)-3-hydroxybutyrate biosynthesis and chemotaxis. Among the inducers hitherto identified, acetoacetate is the classical activator. The AtoSC TCS functional modulation by polyamines, histamine and Ca2+, as well as the role of AtoC as transcriptional regulator, add new promising perspectives in the physiological significance and potential pharmacological exploitation of this TCS in cell proliferation, bacteria–host interactions, chemotaxis, and adaptation.
Keywords: Antizyme; AtoSC two-component system; Escherichia coli ; Histamine; Poly-(R)-3-hydroxybutyrate; Polyamines
Evolution of prokaryotic two-component systems: insights from comparative genomics by David E. Whitworth; Peter J. A. Cock (pp. 459-466).
Two-component systems (TCSs) are diverse and abundant signal transduction pathways found predominantly in prokaryotes. This review focuses on insights into TCS evolution made possible by the sequencing of whole prokaryotic genomes. Typical TCSs comprise an autophosphorylating protein (a histidine kinase), which transfers a phosphoryl group onto an effector protein (a response regulator), thus modulating its activity. Histidine kinases and response regulators are usually found encoded as pairs of adjacent genes within a genome, with multiple examples in most prokaryotes. Recent studies have shed light on major themes of TCS evolution, including gene duplication, gene gain/loss, gene fusion/fission, domain gain/loss, domain shuffling and the emergence of complexity. Coupled with an understanding of the structural and biophysical properties of many TCS proteins, it has become increasingly possible to draw inferences regarding the functional consequences of such evolutionary changes. In turn, this increase in understanding has the potential to enhance both our ability to rationally engineer TCSs, and also allow us to more powerfully correlate TCS evolution with behavioural phenotypes and ecological niche occupancy.
Keywords: Response regulator; Histidine kinase; Hybrid kinase; Gene fusion; Gene fission; Evolution; Recombination; Duplication; Domain
How are signals transduced across the cytoplasmic membrane? Transport proteins as transmitter of information by Larissa Tetsch; Kirsten Jung (pp. 467-477).
In order to adapt to ever changing environmental conditions, bacteria sense environmental stimuli, and convert them into signals that are transduced intracellularly. Several mechanisms have evolved by which receptors transmit signals across the cytoplasmic membrane. Stimulus perception may trigger receptor dimerization and/or conformational changes. Another mechanism involves the proteolytic procession of a receptor whereby a diffusible cytoplasmic protein is generated. Finally, there is increasing evidence that transport proteins play an important role in transducing signals across the membrane. Transport proteins either directly translocate signaling molecules into the cytoplasm, or transmit information via conformational changes to their interacting partners such as membrane-integrated or soluble components of signal transduction cascades. Employing transport proteins as sensors and regulators of signal transduction represents a sophisticated way of interconnecting metabolic flux and transcriptional regulation in cells.
Keywords: Trigger enzymes; ToxR; Histidine kinase; Secondary transporter; ABC transporter
The three-component signalling system HbpS–SenS–SenR as an example of a redox sensing pathway in bacteria by Darío Ortiz de Orué Lucana; Matthew R. Groves (pp. 479-486).
The two-component system SenS–SenR and the extracellular HbpS protein of the cellulose degrader Streptomyces reticuli have been shown to act in concert as a novel system which detects redox stress. In vivo and in vitro experiments have led to the hypothesis that HbpS binds and degrades heme, communicating the extracellular presence of heme and oxidative stress to the membrane-embedded sensor histidine kinase SenS via a bound iron. The response regulator SenR would then up-regulate downstream signalling cascades, leading to the appropriate gene expression levels for bacterial survival in an oxidative environment. Sequence analysis has shown that homologs of HbpS and SenS–SenR exist in a number of ecologically and medically relevant bacterial species, suggesting the existence of a previously undescribed bacterial oxidative stress-response pathway common to both Gram-negative and Gram-positive bacteria. The presented report reviews the current knowledge of the function of this novel protein family consisting of an accessory protein and its cognate two-component system, which could be more properly described as a three-component system.
Osmosensing and osmosignaling in Corynebacterium glutamicum by Reinhard Krämer (pp. 487-497).
The Gram-positive soil bacterium Corynebacterium glutamicum is used in microbial biotechnology for the large-scale production of amino acids, e.g., l-glutamate and l-lysine. We have studied the response of this organism to hyperosmotic challenge at the level of both transcription and protein activity. Two systems responding to hyperosmotic stress in C. glutamicum are reviewed here, the two component system MtrAB and the glycine-betaine uptake system BetP. The osmosensory two-component system consists of the membrane-bound histidine kinase MtrB and the soluble response regulator MtrA. MtrB was shown to perceive a so far unknown physical stimulus related to hyperosmotic stress via the cytoplasmically oriented phosphorylation domain, and to transduce the signal to the DNA via MtrA. The secondary active transporter BetP takes up betaine in cotransport with two Na+ ions. BetP responds to hyperosmotic stress by increased transcription mediated via MtrAB signaling, and by instant activation of transport. In the mechanism of BetP activation, the C-terminal, regulatory domain of BetP, the cytoplasmic concentration of K+, and negative membrane surface charges are involved. The molecular mechanism of the activation process is discussed in relation to the recently published X-ray structure of BetP.
Keywords: Stimulus sensing; Signal transduction; Osmoregulation; Two component system; Transport; Membrane
Tyrosine-kinases in bacteria: from a matter of controversy to the status of key regulatory enzymes by Emmanuelle Bechet; Sébastien Guiral; Sophie Torres; Ivan Mijakovic; Alain-Jean Cozzone; Christophe Grangeasse (pp. 499-507).
When considering protein phosphorylation in bacteria, phosphorylation of aspartic acid and histidine residues mediated by the two-component systems is the first to spring to mind. And yet other phosphorylation systems have been described in bacteria in the past 20 years including eukaryotic-like serine/threonine kinases and more recently tyrosine-kinases. Among the latter, a peculiar type is widespread among bacteria, but not in higher organisms. These enzymes possess unique structural features defining thus a new family of enzymes termed Bacterial tyrosine kinases (BY-kinases). BY-kinases have been shown to be mainly involved in polysaccharide production, but their ability to phosphorylate endogenous substrates indicates that they participate in the regulation of other functions of the bacterial cell. Recent advances in mass spectrometry based phosphoproteomics provided lists of many new phosphotyrosine-proteins, indicating that BY-kinases may be involved in regulating a large array of other cellular functions. One may expect that in a near future, tyrosine phosphorylation will turn out to be one of the key regulatory processes in the bacterial cell and will yield new insights into the understanding of its physiology.
Keywords: Tyrosine-kinase; Phosphorylation; BY-kinase; Bacterial physiology; Regulatory process; Phosphoproteomics; Signaling
Tumor-targeting amino acid auxotrophic Salmonella typhimurium by Robert M. Hoffman (pp. 509-521).
We have developed an effective bacterial cancer therapy strategy by targeting viable tumor tissue using Salmonella typhimurium auxotrophs that we have generated which grow in viable as well as necrotic areas of tumors. However, the auxotrophy severely restricts growth of these bacteria in normal tissue. The S. typhimurium A1-R mutant, which is auxotrophic for leu-arg and has high anti-tumor virulence, was developed in our laboratory. In vitro, A1-R infects tumor cells and causes nuclear destruction. A1-R was initially used to treat metastatic human prostate and breast tumors that had been orthotopically implanted in nude mice. Forty percent of treated mice were cured completely and survived as long as non-tumor-bearing mice. A1-R administered i.v. to nude mice with primary osteosarcoma and lung metastasis was highly effective, especially against metastasis. A1-R was also targeted to both axillary lymph and popliteal lymph node metastasis of human pancreatic cancer and fibrosarcoma, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. The bacteria were delivered via a lymphatic channel to target the lymph node metastases and systemically via the tail vein to target the lung metastasis. The metastases were cured without the need of chemotherapy or any other treatment. A1-R was administered intratumorally to nude mice with an orthotopically transplanted human pancreatic tumor. The primary pancreatic cancer regressed without additional chemotherapy or any other treatment. A1-R was also effective against pancreatic cancer liver metastasis when administered intrasplenically to nude mice. The approach described here, where bacterial monotherapy effectively treats primary and metastatic tumors, is a significant improvement over previous bacterial tumor-therapy strategies that require combination with toxic chemotherapy. Three promoter clones engineered in S. enterica typhimurium were identified to have enhanced expression in bacteria growing in tumors relative to those growing in the spleen. The expression of therapeutics in Salmonella under the regulation of one or more promoters that are activated preferentially in tumors has the potential to improve the efficacy of Salmonella tumor therapy. Exploitation of the tumor-killing capability of Salmonella has great promise for a new paradigm of cancer therapy.
Keywords: S. typhimurium ; Leucine-arginine auxotrophs; Cancer therapy; Green fluorescent protein; Red fluorescent protein
Site-directed mutagenesis and computational study of the Y366 active site in Bacillus subtilis protoporphyrinogen oxidase by Lu Sun; Xin Wen; Ying Tan; Heyang Li; Xing Yang; Yuefang Zhao; Baifan Wang; Qiongyao Cao; Congwei Niu; Zhen Xi (pp. 523-530).
Protoporphyrinogen IX oxidase (PPO), the last common enzyme of heme and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX, with FAD as cofactor. Among PPO, Bacillus subtilis PPO (bsPPO) is unique because of its broad substrate specificity and resistance to inhibition by diphenylethers. Identification of the activity of bsPPO would help us to understand the catalysis and resistance mechanisms. Based on the modeling and docking studies, we found that Y366 site in bsPPO was adjacent to substrate and FAD. In order to evaluate the functional role of this site, three mutants Y366A Y366E and Y366H were cloned and kinetically characterized. The efficiency of catalysis for Y366A and Y366H reduced to 10% of the wild-type enzyme’s activity, while Y366E just retained 1%. Y366E shows large resistance (K i = 153.94 μM) to acifluorfen. Molecular docking was carried out to understand the structure and functional relationship of PPO. The experimental results from the site-directed mutagenesis are consistent with the computational studies. The residue at position 366 is seemed to be responsible for substrate binding and catalysis and involved in herbicide resistance of bsPPO.
Keywords: Kinetics; Molecular docking; Protoporphyrinogen oxidase; Site-directed mutagenesis
The novel inhibitors of serine proteases by N. Hovhannisyan; Sh. Harutyunyan; A. Hovhannisyan; A. Hambardzumyan; M. Chitchyan; M. Melkumyan; G. Oganezova; N. Avetisyan (pp. 531-536).
Thirty optically active nonprotein α-amino acids and peptides based thereon have been screened for their ability to interact with bovine trypsin and proteinase K from Tritirachium album Limber, which belong to the group of serine proteases. Both structure-based drug design approach and determination of enzyme activity have been used to identify low molecular weight inhibitors of trypsin and proteinase K. Compounds have been selected that according to the docking analysis were able to interact with trypsin and proteinase K. Following the docking analysis measurement of enzymes activity (2R,3S)-β-hydroxyleucine and (2S,3R)-β-hydroxyleucine inhibited both enzymes activity, whereas (S)-α-methyl-β-phenylalanine, (R)-α-methyl-β-phenylalanine, (S)-allylglycine, (R)-allylglycine, (S)-α-allylalanine, (R)-α-allylalanine and allo-O-ethylthreonine inhibited only proteinase K; and N-formyl-(S)-methionyl-(2S,3R)-hydroxyleucine, N-formyl-(S)-methionyl-(2R,3S)-hydroxyleucine, N-formyl-(S)-methionyl-(S)-allylglycine and N-formyl-(S)-methionyl-(R)-allylglycine inhibited trypsin. It has been shown that inhibition of trypsin by (2R,3S)-β-hydroxyleucine and N-formyl-(S)-methionyl-(2R,3S)-hydroxyleucine is of a competitive mode.
Keywords: Trypsin; Proteinase K; Nonprotein amino acids; Peptides; Inhibitors; Docking
Synthesis and characterization of new Pd(II) complexes of l-ethylphenylalanate by Abdol R. Hajipour; Kazem Karami; Azadeh Pirisedigh; Arnold E. Ruoho (pp. 537-541).
Complex (S,S)-[Pd{C6H4(CH2CHNH2CO2CH2CH3)}(μ-Br)]2 (3) was prepared following the method by Vicente and Saura-Llamas (Organometallics 26:2768–2776, 2007), by the reaction of l-ethylphenylalanate and Pd(OAc)2 in 1:1 molar ratio under acetonitrile heating conditions and subsequently treating with NaBr. In addition, the cleavage of halogeno-bridge of the complex 3 via nucleophilic attack of some neutral ligands such as triphenylphosphine, pyridine, 2,4,6-trimethylpyridine and piperidine were investigated and the corresponding complexes (S)-[Pd{C6H4(CH2CHNH2CO2CH2CH3)(Y)(Br)}] (4a–f) were obtained in moderate yields. The six-member orthopalladated complexes were characterized by 1H-NMR, 31P-NMR, FT-IR and elemental analysis techniques.
Keywords: Phenylalanine; Cyclopalladation; Orthometallate
Characterization of prolidase I and II purified from normal human erythrocytes: comparison with prolidase in erythrocytes from a patient with prolidase deficiency by Soichiro Uramatsu; Gang Liu; Qing Yang; Mutsumi Uramatsu; Haidong Chi; Jincai Lu; Koichi Yamashita; Hiroyuki Kodama (pp. 543-551).
The effect of various sulfur-containing amino acids on the activities of prolidase isoenzymes I and II isolated from erythrocytes of healthy individuals, and erythrocyte lysates from a patient with prolidase deficiency was investigated. The activity of prolidase I against glycylproline was strongly enhanced by d-methionine. l-Methionine and d,l-methionine slightly enhanced the activity at low concentration, but N-acetyl-l-methionine had no effect. d-Ethionine, l-ethionine, and d,l-ethionine also enhanced the activity of prolidase I. d,l-Homocysteine enhanced the activity at low concentration, but inhibited the activity at 50 mM. The activity of prolidase II against methionylproline was enhanced by d-methionine, d,l-methionine, and l-methionine, but N-acetyl-l-methionine had no effect. d-Ethionine and d,l-ethionine strongly enhanced the activity of prolidase II compared with l-ethionine; d,l-homocysteine weakly enhanced the activity. d,l-Homocysteine-thiolactone inhibited the activities of prolidase I and II in a concentration-dependent manner. The effect of various sulfur-containing amino acids on prolidase activity against methionylproline in erythrocyte lysates from a patient with prolidase deficiency was almost the same as that on prolidase II. The kinetics of the activities of prolidase I, II, and patient prolidase were also studied. Their K m values were changed by adding sulfur-containing amino acids, but V max values were unchanged.
Keywords: Sulfur-containing amino acids; Prolidase isoenzymes; Prolidase deficiency; Erythrocytes
How are signals transduced across the cytoplasmic membrane? Transport proteins as transmitter of information
by Larissa Tetsch; Kirsten Jung (pp. 553-554).