Ligand binding specificity of the Escherichia coli periplasmic histidine binding protein,HisJ

The HisJ protein from Escherichia coli and related Gram negative bacteria is the periplasmic component of a bacterial ATP‐cassette (ABC) transporter system. Together these proteins form a transmembrane complex that can take up L‐histidine from the environment and translocate it into the cytosol. We have studied the specificity of HisJ for binding L‐His and many related naturally occurring compounds. Our data confirm that L‐His is the preferred ligand, but that 1‐methyl‐L‐His and 3‐methyl‐L‐His can also bind, while the dipeptide carnosine binds weakly and D‐histidine and the histidine degradation products, histamine, urocanic acid and imidazole do not bind. L‐Arg, homo‐L‐Arg, and post‐translationally modified methylated Arg‐analogs also bind with reasonable avidity, with the exception of symmetric dimethylated‐L‐Arg. In contrast, L‐Lys and L‐Orn have considerably weaker interactions with HisJ and methylated and acetylated Lys variants show relatively poor binding. It was also observed that the carboxylate group of these amino acids and their variants was very important for proper recognition of the ligand. Taken together our results are a key step towards designing HisJ as a specific protein‐based reagentless biosensor.     

Altering the binding activity and specificity of the leucine binding proteins of Escherichia coli

Two leucine-binding proteins with overlapping specificities for the branched-chain amino acids are present in Escherichia coli. In order to study the basis of specificity for the very similar hydrophobic ligands, we have constructed a series of site-directed mutants of both proteins based on inspection of the leucine-isoleucine-valine-binding protein crystal structure reported by Sack et al. (Sack, J. S., Saper, M. A., and Quiocho, F. A. (1989) J. Mol. Biol. 206, 171-191). Each of the mutant proteins was overexpressed and purified, and their binding activity for a wide variety of potential ligands was measured. By introducing a common restriction endonuclease cleavage site in the two proteins, two hybrid binding proteins consisting of the amino-terminal third of one binding protein fused to the carboxyl-terminal two-thirds of the other were created. The results of these studies indicated that the binding site of the leucine-isoleucine-valine binding protein can accommodate a branch at the beta-carbon of the ligand and that hydrophilic groups on the ligand can be accommodated only in certain orientations. None of the single amino acid substitutions resulted in complete switches in specificity between the two proteins, suggesting that additional residues are involved in leucine binding and discrimination among the branched-chain amino acid substrates.     

DNA binding of Escherichia coli arginine repressor mutants altered in oligomeric state

The Escherichia coli arginine repressor (ArgR) controls expression of the arginine biosynthetic genes and acts as an accessory protein in Xer site-specific recombination at cer and related plasmid recombination sites. The hexameric wild-type protein shows L -arginine-dependent DNA binding. In this work, ArgR mutants that are defective in trimer–trimer interactions and bind DNA as trimers in an L -arginine-independent manner are isolated and characterized. Whereas the wild-type ArgR hexamer exhibits high-affinity binding to two repeated ARG boxes separated by 3 bp (each ARG box containing two identical dyad symmetrical 9 bp half-sites), the trimeric mutants bind to and footprint three adjacent half-sites of this 'idealized' substrate. Trimeric ArgR is impaired in its ability to repress the arginine biosynthetic genes and in Xer site-specific recombination. In the absence of L -arginine, residual wild-type ArgR-binding occurs as trimers. The binding of an N-terminal 77-amino-acid DNA-binding domain to idealized ARG boxes is also characterized.     

Quantitative analysis of DNA binding by the Escherichia coli arginine repressor

The cis-trans isomerisation of maleylacetoacetate to fumarylacetoacetate is the penultimate step in the tyrosine/phenylalanine catabolic pathway and has recently been shown to be catalysed by glutathione S-transferase enzymes belonging to the zeta class. Given this primary metabolic role it is unsurprising that zeta class glutathione S-transferases are well conserved over a considerable period of evolution, being found in vertebrates, plants, insects and fungi. The structure of this glutathione S-transferase, cloned from Arabidopsis thaliana, has been solved by single isomorphous replacement with anomalous scattering and refined to a final crystallographic R-factor of 19.6% using data from 25.0 A to 1.65 A. The zeta class enzyme adopts the canonical glutathione S-transferase fold and forms a homodimer with each subunit consisting of 221 residues. In agreement with structures of glutathione S-transferases from the theta and phi classes, a serine residue (Ser17) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione. Site-directed mutagenesis of this residue confirms its importance in catalysis. In addition, the role of a highly conserved cysteine residue (Cys19) present in the active site of the zeta class glutathione S-transferase enzymes is discussed.     

Relieving effects of glycine and methionine from acetic acid inhibition in Escherichia coli fermentation

Among amino acids screened for their potential to relieve wild and recombinant Escherichia coli from the negative effects of acetic acid, glycine, and methionine showed a sparing effect. In the presence of 2 g/L of acetic acid, addition of 0.5 g/L of glycine or methionine resulted in either a complete recovery or a further enhancement in the specific growth rate, while the enhancement was significant but not fully complete in the presence of 4 g/L of acetic acid. The addition of 0.5 g/L of methionine alleviated the negative effect of acetic acid on recombinant E. Coli growth to produce more beta-lactamase, which was encoded by plasmid pUC18. In continuous fermentation the methionine effect on recombinant. E. coli metabolism depended on dilution rate; at high dilution rates, above 0.4 h(-1), the methionine addition enhanced beta-lactamase production and reduced acetic acid formation, while at low dilution rates, below 0.3 h (-1), the effect was reversed. In def-batch fermentation with wild-type E. Coli, cell growth rate and cell yield from glucose were enhanced with methionine addition, while the acetic acid concentration reached over 4 g/L. (c) 1993 John Wiley & Sons, Inc.     

Effects of deregulation of methionine biosynthesis on methionine excretion in Escherichia coli

Several regulators of methionine biosynthesis have been reported in Escherichia coli, which might represent barriers to the production of excess l-methionine (Met). In order to examine the effects of these factors on Met biosynthesis and metabolism, deletion mutations of the methionine repressor (metJ) and threonine biosynthetic (thrBC) genes were introduced into the W3110 wild-type strain of E. coli. Mutations of the metK gene encoding S-adenosylmethionine synthetase, which is involved in Met metabolism, were detected in 12 norleucine-resistant mutants. Three of the mutations in the metK structural gene were then introduced into metJ and thrBC double-mutant strains; one of the resultant strains was found to accumulate 0.13 g/liter Met. Mutations of the metA gene encoding homoserine succinyltransferase were detected in alpha-methylmethionine-resistant mutants, and these mutations were found to encode feedback-resistant enzymes in a 14C-labeled homoserine assay. Three metA mutations were introduced, using expression plasmids, into an E. coli strain that was shown to accumulate 0.24 g/liter Met. Combining mutations that affect the deregulation of Met biosynthesis and metabolism is therefore an effective approach for the production of Met-excreting strains.     

Synthesis and β-conformation of sequential polypeptides containing arginine,histidine, and leucine

Alternating poly(Arg-Leu) and copolypeptides with Arg-Leu and His-Leu sequences were prepared by condensation of the corresponding p-nitrophenyl dipeptide esters in the presence of 1-hydroxybenzotriazole. Arginine was used without any protection and histidine side chains were protected using π-benzyloxymethyl group recently introduced in peptide chemistry. The ability of these polypeptides with alternating hydrophilic and hydrophobic residues to form water-soluble β-sheets was investigated by CD.     

Identification of residues involved in the binding of methionine by Escherichia coli methionyl-tRNA synthetase

Comparison of the amino-acid sequences of several methionyl-tRNA synthetases indicates the occurrence of a few conserved motifs, having a possible functional significance. The role of one of these motifs, centered at position 300 in the E. coli enzyme sequence, was assayed by the use of site-directed mutagenesis. Substitution of the His301 or Trp305 residues by Ala resulted in a large decrease in methionine affinity, whereas the change of Val298 into Ala had only a moderate effect. The catalytic rate of the enzyme was unimpaired by these substitutions. It is concluded that the above conserved amino-acid region is located at or close to the amino-acid binding pocket of methionyl-tRNA synthetase.     

Selective inhibition of synaptosomal proline uptake by leucine and methionine enkephalins

The high affinity, sodium-dependent uptake of proline by rat brain synaptosomes was inhibited by the opioid pentapeptides, Leu-enkephalin and Met-enkephalin. The synaptosomal uptake of other putative neurotransmitter amino acids including glutamic acid, aspartic acid, gamma-aminobutyric acid, and taurine was not altered in the presence of enkephalins. The uptake of a neuroinactive amino acid, leucine, was also unaffected by enkephalins. The extent of proline uptake was half-maximal at a Leu-enkephalin concentration of 1 microM. Both the initial rate of transport and the overall capacity for proline accumulation were reduced. The effect of the enkephalins was vectorial since carrier-mediated efflux of proline was not altered in the presence of enkephalins. Morphine and the opioid peptides, dynorphin and beta-endorphin, were without effect on proline uptake. The inhibition of proline uptake by enkephalins was not diminished by prior incubation of the synaptosomal preparation with naloxone; however, the inhibition was attenuated by 1-butanol. The des-tyrosyl fragments of the enkephalins were as inhibitory as the intact pentapeptides. A modified enkephalin ([D-Ser2]Leu-enkephalin-Thr) with selective affinity for the delta subclass of enkephalin receptor was effective in inhibiting proline uptake. On the basis of the selectivity of these effects, we propose that there is a specific population of nerve endings in the cerebral cortex that contains both a proline-transport system and binding sites for Leu- and Met-enkephalin and furthermore, that these binding sites may be related to the putative delta receptor.     

Regulation of arginine biosynthesis,catabolism and transport in Escherichia coli

Amino Acids - Already very early, the study of microbial arginine biosynthesis and its regulation contributed significantly to the development of new ideas and concepts. Hence, the term...