Involvement of arginine residues in the allosteric activation of Escherichia coli ADP-glucose synthetase

Biochemistry ◽  
1982 ◽  
Vol 21 (8) ◽  
pp. 1929-1934 ◽  
Author(s):  
Curtis A. Carlson ◽  
Jack Preiss
Keyword(s):  
Escherichia Coli ◽  
Allosteric Activation ◽  
Arginine Residues

scholarly journals Genetic Analysis of Pathway Specificity during Posttranslational Protein Translocation across the Escherichia coli Plasma Membrane

2003 ◽  
Vol 185 (9) ◽  
pp. 2811-2819 ◽  
Author(s):  
Natascha Blaudeck ◽  
Peter Kreutzenbeck ◽  
Roland Freudl ◽  
Georg A. Sprenger
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Protein Translocation ◽  
Alternative Possibilities ◽  
Amino Acid Residues ◽  
Sec Pathway ◽  
Twin Arginine Translocation ◽  
Tat Pathway ◽  
Arginine Residues ◽  
Dependent Protein

ABSTRACT In Escherichia coli, the SecB/SecA branch of the Sec pathway and the twin-arginine translocation (Tat) pathway represent two alternative possibilities for posttranslational translocation of proteins across the cytoplasmic membrane. Maintenance of pathway specificity was analyzed using a model precursor consisting of the mature part of the SecB-dependent maltose-binding protein (MalE) fused to the signal peptide of the Tat-dependent TorA protein. The TorA signal peptide selectively and specifically directed MalE into the Tat pathway. The characterization of a spontaneous TorA signal peptide mutant (TorA*), in which the two arginine residues in the c-region had been replaced by one leucine residue, showed that the TorA*-MalE mutant precursor had acquired the ability for efficiently using the SecB/SecA pathway. Despite the lack of the “Sec avoidance signal,” the mutant precursor was still capable of using the Tat pathway, provided that the kinetically favored Sec pathway was blocked. These results show that the h-region of the TorA signal peptide is, in principle, sufficiently hydrophobic for Sec-dependent protein translocation, and therefore, the positively charged amino acid residues in the c-region represent a major determinant for Tat pathway specificity. Tat-dependent export of TorA-MalE was significantly slower in the presence of SecB than in its absence, showing that SecB can bind to this precursor despite the presence of the Sec avoidance signal in the c-region of the TorA signal peptide, strongly suggesting that the function of the Sec avoidance signal is not the prevention of SecB binding; rather, it must be exerted at a later step in the Sec pathway.

scholarly journals Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

2002 ◽  
Vol 365 (1) ◽  
pp. 303-309 ◽  
Author(s):  
Wynand B.L. ALKEMA ◽  
Antoon K. PRINS ◽  
Erik de VRIES ◽  
Dick B. JANSSEN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Penicillin Acylase ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

scholarly journals NleB2 from enteropathogenic Escherichia coli is a novel arginine-glucose transferase effector

2021 ◽  
Vol 17 (6) ◽  
pp. e1009658
Author(s):  
Cristina Giogha ◽  
Nichollas E. Scott ◽  
Tania Wong Fok Lung ◽  
Georgina L. Pollock ◽  
Marina Harper ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Host Cell ◽  
Peptide Sequencing ◽  
Effector Proteins ◽  
Host Protein ◽  
Death Domain ◽  
T3ss Effector ◽  
Arginine Residues

During infection, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) directly manipulate various aspects of host cell function through the translocation of type III secretion system (T3SS) effector proteins directly into the host cell. Many T3SS effector proteins are enzymes that mediate post-translational modifications of host proteins, such as the glycosyltransferase NleB1, which transfers a single N-acetylglucosamine (GlcNAc) to arginine residues, creating an Arg-GlcNAc linkage. NleB1 glycosylates death-domain containing proteins including FADD, TRADD and RIPK1 to block host cell death. The NleB1 paralogue, NleB2, is found in many EPEC and EHEC strains but to date its enzymatic activity has not been described. Using in vitro glycosylation assays combined with mass spectrometry, we found that NleB2 can utilize multiple sugar donors including UDP-glucose, UDP-GlcNAc and UDP-galactose during glycosylation of the death domain protein, RIPK1. Sugar donor competition assays demonstrated that UDP-glucose was the preferred substrate of NleB2 and peptide sequencing identified the glycosylation site within RIPK1 as Arg603, indicating that NleB2 catalyses arginine glucosylation. We also confirmed that NleB2 catalysed arginine-hexose modification of Flag-RIPK1 during infection of HEK293T cells with EPEC E2348/69. Using site-directed mutagenesis and in vitro glycosylation assays, we identified that residue Ser252 in NleB2 contributes to the specificity of this distinct catalytic activity. Substitution of Ser252 in NleB2 to Gly, or substitution of the corresponding Gly255 in NleB1 to Ser switches sugar donor preference between UDP-GlcNAc and UDP-glucose. However, this switch did not affect the ability of the NleB variants to inhibit inflammatory or cell death signalling during HeLa cell transfection or EPEC infection. NleB2 is thus the first identified bacterial Arg-glucose transferase that, similar to the NleB1 Arg-GlcNAc transferase, inhibits host protein function by arginine glycosylation.

scholarly journals Why Does Escherichia coli Grow More Slowly on Glucosamine than on N-Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

2005 ◽  
Vol 187 (9) ◽  
pp. 2974-2982 ◽  
Author(s):  
Laura I. Álvarez-Añorve ◽  
Mario L. Calcagno ◽  
Jacqueline Plumbridge
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Sugar Transport ◽  
Point Mutations ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Allosteric Activation

ABSTRACT Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

Identification of Two Essential Arginine Residues in UhpT, the Sugar Phosphate Antiporter of Escherichia coli

1998 ◽  
Vol 164 (2) ◽  
pp. 187-195 ◽  
Author(s):  
M.-C. Fann ◽  
A.H. Davies ◽  
A. Varadhachary ◽  
T. Kuroda ◽  
C. Sevier ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sugar Phosphate ◽  
Arginine Residues
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