Temperature-sensitive glutamate dehydrogenase mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium defective in glutamate dehydrogenase activity were isolated in parent strains lacking glutamate synthase activity by localizcd mutagenesis or by a general mutagenesis combined with a cycloserine enrichment for glutamate auxotrophs. Two mutants with temperature-sensitive phenotypes had glutamate dehydrogenase activities that were more thermolabile than that of an isogenic control strain. Eight other mutants had less than 10% of the wild-type glutamate dehydrogenase activity. All the mutations were cotransducible with a Tn10 element (zed-2:Tn10) located at approximately 23 U on the S. typhimurium linkage map. These data strongly indicate that this region contains the structural gene (gdhA) for glutamate dehydrogenase.     

Effect of mutation of lysine-120, located at the entry to the active site of O-acetylserine sulfhydrylase-A from Salmonella typhimurium

O-Acetylserine sulfhydrylase catalyzes the final step of the biosynthesis of L-cysteine, the replacement of the beta-acetoxy group of O-acetyl-L-serine (OAS) by a thiol. The enzyme undergoes a conformational change to close the site upon formation of the external Schiff base (ESB) with OAS. Mutation of K120 to Q was predicted to destabilize the closed form of the ESB and decrease the rate. The K120Q mutant enzyme was prepared and characterized by UV-visible absorbance, fluorescence, visible CD, and 31P NMR spectral studies, as well as steady state and pre-steady state kinetic studies. Spectra suggest a shift in the tautomeric equilibrium toward the neutral enolimine and an increase in the rate of interconversion of the open and closed forms of the enzyme. A decrease in the rate of both half reactions likely reflects the stabilization of the ESB as a result of the increased rate of equilibration of the open and closed forms of the enzyme along the reaction pathway. Data suggest a role of K120 in helping to stabilize the closed conformation by participating in a new hydrogen bond to the backbone carbonyl of A231.     

Kinetic studies of a NADP+-specific isocitrate dehydrogenase from Salmonella typhimurium. Purification and reaction mechanism

The kinetics of a Mn²⁺-requiring, NADP⁺-specific isocitrate dehydrogenase from Salmonella typhimurium have been examined by the measurement of initial velocity rates in the presence and absence of the reaction products. The binding of each of the cosubstrates, isocitrate, and NADP⁺, is not independent of the other, and the isocitrate-Mn²⁺ complex is the kinetically important substrate species. All of the reaction products, α-ketoglutarate, CO₂, and NADPH are competitive with both cosubstrates and the mechanism appears to be of the rapid equilibrium random type. The enzyme has been purified to homogeneity and has an isoelectric point at pH 4.0–4.2, and an apparent molecular weight of 102,000.     

Genetic characterization of the glutamate dehydrogenase gene (gdhA) of Salmonella typhimurium.

Salmonella typhimurium mutants, either devoid or glutamate dehydrogenase activity or having a thermolabile glutamate dehydrogenase protein, were used to identify the structural gene (gdhA) for this enzyme. Transductions showed that the mutations producing these phenotypes were linked to both the pncA and nit genes, placing the gdhA locus between 23 and 30 U on the S. typhimurium chromosome. Additional transductions with several Tn10 insertions established the gene order as pncA-gdhA-nit. Since few genetic markers exist in this region of the chromosome, Hfr strains were constructed to orient the pncA-gdhA-nit cluster with outside genes. Conjugation experiments provided evidence for the gene order pyrD-pncA-gdhA-nit-trp. To further characterize gdhA, we used Mu cts d1 (Apr lac) insertions in this gene to select numerous strains containing deletions with various endpoints. Transductions of these deletions with strains containing different gdh mutations and with a mutant having a thermolabile glutamate dehydrogenase protein permitted us to construct a deletion map of the gdhA region.     

Cloning and characterization of gdhA, the structural gene for glutamate dehydrogenase of Salmonella typhimurium.

Glutamic acid is synthesized in enteric bacteria by either glutamate dehydrogenase or by the coupled activities of glutamate synthase and glutamine synthetase. A hybrid plasmid containing a fragment of the Salmonella typhimurium chromosome cloned into pBR328 restores growth of glutamate auxotrophs of S. typhimurium and Escherichia coli strains which have mutations in the genes for glutamate dehydrogenase and glutamate synthase. A 2.2-kilobase pair region was shown by complementation analysis, enzyme activity measurements, and the maxicell protein synthesizing system to carry the entire glutamate dehydrogenase structural gene, gdhA. Glutamate dehydrogenase encoded by gdhA carried on recombinant plasmids was elevated 5- to over 100-fold in S. typhimurium or E. coli cells and was regulated in both organisms. The gdhA promoter was located by recombination studies and by the in vitro fusion to, and activation of, a promoter-deficient galK gene. Additionally, S. typhimurium gdhA DNA was shown to hybridize to single restriction fragments of chromosomes from other enteric bacteria and from Saccharomyces cerevisiae.     

Crystal structure of the lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium at 2.7-A resolution.

A wide variety of sugars, amino acids, peptides, and inorganic ions are transported into bacteria by periplasmic transport systems consisting of substrate-specific receptors (binding proteins) and membrane-bound protein complexes. The crystal structure of the lysine-, arginine-, ornithine-binding protein (LAO) at 2.7-A resolution shows that the molecule has a bi-lobal structure and that its topological structure is different from other amino acid-binding proteins but is similar to the sulfate-binding protein and maltose-binding protein. High sequence homology between LAO and the histidine-binding protein (HisJ) and the fact that LAO and HisJ share the same membrane-bound protein complex allow one to define functional regions responsible for the ligand binding and for the interaction with the membrane complex.     

The catalytically active form of histidinol dehydrogenase from Salmonella typhimurium.

The active-enzyme-sedimentation procedure was used to identify the catalytically competent form of histidinol dehydrogenase (EC 1.1.1.23) isolated from Salmonella typhimurium. At pH 9.4 the active species has a sedimentation coefficient S20,W of 5.4S, indicating that the dimer with a mol.wt. of approx. 83 000 is the enzymically active form.     

L-Histidinol phosphate aminotransferase from Salmonella typhimurium. Kinetic behavior and sequence at the pyridoxal-P binding site

A coupled assay with alpha-hydroxyglutarate dehydrogenase was used to analyze the kinetic behavior of histidinol phosphate aminotransferase from Salmonella typhymurium. Data obtained from studies of initial velocity, inhibition by products or substrate analogues, isotope exchange rates, and the determination of the equilibrium constant were consistent only with a Ping-Pong Bi Bi mechanism. Variations in inhibition patterns by different substrate analogues indicate that the microenvironment about the pyridoxal phosphate and the pyridoxamine phosphate forms of histidinol phosphate amino-transferase are different, and favor the presence of one active site with partially overlapping substrate-binding subsites for these 2 forms of the enzyme. Histidinol phosphate aminotransferase also catalyzes decomposition of beta-chloro-L-alanine to pyruvate, NH3 and Cl-; no transamination of this substrate occurs and inactivation of the enzyme accompanies this reaction. After reduction of histidinol-P aminotransferase with [3H]NaBH4, carboxymethylation, and tryptic digestion, one major radioactive peptide absorbing at 325 nm was isolated. Its primary structure was determined to be TLSK*AFALAGLR, where K* is the P-pyridoxyllysine residue. Although this peptide is only 30-40% homologous with the corresponding segment reported for other transaminases, all of these peptides are similar in placement of an hydroxyamino acid residue three residues upstream from the lysine residue, and in the cluster of hydrophobic amino acid residues immediately following the lysine residue.     

Salmonella typhimurium histidinol dehydrogenase: complete reaction stereochemistry and active site mapping

The stereochemistry of the L-histidinol dehydrogenase reaction was determined to be R at NAD for both steps, confirming previous results with a fungal extract [Davies, D., Teixeira, A., & Kenworthy, P. (1972) Biochem. J. 127, 335-343]. NMR analysis of monodeuteriohistidinols produced by histidinol/NADH exchange reactions arising via reversal of the alcohol oxidation reaction indicated a single stereochemistry at histidinol for that step. Comparison of vicinal coupling values of the exchange products with those of L-alaninol and a series of (S)-2-amino-1-alcohols allowed identification of the absolute stereochemistry of monodeuteriohistidinols and showed that histidinol dehydrogenase removes first the pro-S then the pro-R hydrogens of substrate histidinol. The enzyme stereochemistry was confirmed by isotope effects for monodeuteriohistidinols as substrates for the pro-R-specific dehydrogenation catalyzed by liver alcohol dehydrogenase. Active site mapping was undertaken to investigate substrate-protein interactions elsewhere in the histidinol binding site. Critical binding regions are the side-chain amino group and the imidazole ring, whose methylation at the 1- or 2-position caused severe decreases in binding affinity. Use of alternative substrates further clarified active site interactions with the substrate. Compounds in which the alpha-amino group was replaced by chloro, bromo, or hydrogen substituents were not substrates of the overall reaction at 1/10,000 the normal rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae

The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentrations increases. A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7.5. Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization. The following results suggest that the decrease in NADP-GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls.     

Purification and characterization of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium.

The lysine-, arginine-, ornithine-binding protein (LAO) from Salmonella typhimurium has been purified to homogeneity and characterized. The dissociation constants (KD) were determined by equilibrium dialysis assay to be 14, 15, and 29 nM for L-arginine, L-lysine, and L-ornithine respectively. L-Histidine was found to be a relatively good ligand (KD, 500 nM). Methods have been developed for the separation of liganded from unliganded LAO, for the estimation of bound ligand, and for unliganding LAO. Liganded and unliganded LAO are shown to have distinct UV spectra. The UV spectrum also varies with the nature of the substrate. Inhibition studies with substrate analogs yielded information useful for understanding the nature of the ligand-binding pocket.     

Active site plasticity of endonuclease V from Salmonella typhimurium

Base deamination is a major type of DNA damage under nitrosative stress. Endonuclease V initiates repair of deaminated base damage by making a nucleolytic incision one nucleotide away from the 3' side of the lesion. Within the endonuclease V family, the substrate specificities are different from one enzyme to another. In this study, we investigated deamination lesion cleavage activities of endonuclease V from the macrophage-residing pathogen, Salmonella typhimurium. Salmonella endonuclease V exhibits limited turnover on cleavage of deoxyinosine- and xanthosine-containing DNA. Binding analysis indicates that this single-turnover property is caused by tight binding to nicked products. The nicking activity is similar between the double-stranded deoxyinosine- and deoxyxanthosine-containing DNA. Cleavage rates are not affected by bases opposite the deoxyinosine or deoxyxanthosine lesions. The enzyme is also active on single-stranded deoxyinosine- and deoxyxanthosine-containing DNA. Unlike endonuclease V from Thermotoga maritima, Salmonella endonucleae V can only turnover deoxyuridine-containing DNA to a limited extent when substrate is in excess. Binding analysis indicates that Salmonella endonuclease V achieves tight binding to deoxyuridine-containing DNA, a property that distinguishes it from Thermotoga endonuclease V. Cleavage analysis on mismatch-containing DNA also indicates that the active site of Salmonella endonuclease V can accommodate pyrimidine-containing mismatches, resulting in more comparable cleavage of pyrimidine- and purine-containing mismatches. This comprehensive DNA cleavage and binding analysis reveals the plastic nature in the active site of Salmonella endonuclease V, which allows the enzyme to enfold both purine and pyrimidine deaminated lesions or base pair mismatches.     

Reaction of the nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 2',5'-bisphosphate at the coenzyme site of wild-type and mutant NADP(+)-specific glutamate dehydrogenases from Salmonella typhimurium.

Wild-type glutamate dehydrogenase (EC 1.4.1.4) from Salmonella typhimurium reacts at 25 degrees C in 0.1 M phosphate buffer, pH 7, with the nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 2',5'-bisphosphate (2-BDB-TA 2',5'-DP) to give 78% inactivation. Protection against inactivation was achieved with NADPH, indicating that modification occurred in the region of the coenzyme binding site. After reaction of the enzyme with 2-BDB-TA 2',5'-DP, the dioxo moiety of the bound reagent was reduced with [3H]NaBH4. The radioactive peptide which corresponds to the sequence Leu282-Cys283-Glu284-Ile285-Lys286 was isolated by HPLC from tryptic digests of inactive modified enzyme but was absent in digests of active enzyme modified in the presence of NADPH. Mutant enzyme E284Q was 64% inactived by 2-BDB-TA 2',5'-DP and modification of the corresponding Leu282-Lys286 peptide was found, while neither mutant enzyme C283I nor C283I:E284Q was inactivated by the nucleotide analogue and no corresponding radioactive peptides were found. These results show that cysteine-283 is the target of the reagent and is located near the coenzyme binding site. The nucleotide analogue 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A 2',5'-DP) has also been shown to react with cysteine-283 (L. Haeffner-Gormley et al., 1991, J. Biol. Chem. 266, 5388-5394). However, the predominant form of the Leu282-Lys286 peptide after reaction with 2-BDB-TA 2',5'-DP contained only 0.17 mol tritium/mol leucine, whereas the 2-BDB-T epsilon A 2',5'-DP-modified peptide contained 1.80 mol tritium/mol leucine; these results indicate that the reaction product of 2-BDB-T epsilon A 2',5'-DP retains two reducible carbonyl groups while these are not available in the product of 2-BDB-TA 2',5'-DP. It is suggested that cysteine-283 reacts primarily at a carbonyl group of 2-BDB-TA 2',5'-DP to form a thiohemiacetal derivative, while it reacts at the methylene group of 2-BDB-T epsilon A 2',5'-DP with displacement of bromide. Both nucleotide analogues also yielded, in small amount, a crosslinked peptide containing the sequences 282-286 and 299-333, indicating proximity between these regions in the native structure.     

Properties of phosphorylated NADP+-specific glutamate dehydrogenase fromEscherichia coli

The NADP⁺-specific glutamate dehydrogenase (GDH) fromEscherichia coli strain D₅H₃G₇, an enzyme that catalyzes the interconversion of -ketoglutarate andl-glutamate, has been shown to be phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein is extremely acid labile and is unstable at high pH. Treatment of GDH with diethyl pyrocarbonate (DEP), a histidine-modifying reagent, blocked the incorporation of³²P from [-³²P]ATP. GDH catalytic activity was also inhibited by DEP treatment. Hydroxylamine, a reagent hydrolyzing phosphoramidates, catalyzed the removal of phosphate from phosphorylated GDH, suggesting that GDH may be phosphorylated at a histidine residue(s). A total enzymatic hydrolysis of phosphorylated GDH, which was electroeluted from a native polyacrylamide gel, was analyzed by a Dowex 1-8X anion exchange chromatography. The presence of³²P-labeled 3-phosphohistidine, characterized and identified from this hydrolysate, demonstrates that a histidine residue(s) is the site of phosphorylation.     

In vivo phosphorylation of NADP+ glutamate dehydrogenase inEscherichia coli

The NADP⁺-specific glutamate dehydrogenase inEscherichia coli K12 has been shown to be phosphorylated in vivo when the cells are grown in a low-phosphate minimal salts medium containing³²P inorganic phosphate. The amount of radioactivity incorporated into the enzyme is different depending on the growth phase of the culture, with the highest level of³²P incorporation occurring during the mid-exponential growth phase. Previously reported studies have demonstrated that the enzyme is also phosphorylated in vitro in an ATP-dependent reaction.     

Allosteric discrimination at the NADH/ADP regulatory site of glutamate dehydrogenase

Glutamate dehydrogenase (GDH) is a target for treating insulin‐related disorders, such as hyperinsulinism hyperammonemia syndrome. Modeling native ligand binding has shown promise in designing GDH inhibitors and activators. Our computational investigation of the nicotinamide adenine diphosphate hydride (NADH)/adenosine diphosphate (ADP) site presented in this paper provides insight into the opposite allosteric effects induced at a single site of binding inhibitor NADH versus activator ADP to GDH. The computed binding free‐energy difference between NADH and ADP using thermodynamic integration is ?0.3 kcal/mol, which is within the ?0.275 and ?1.7 kcal/mol experimental binding free‐energy difference range. Our simulations show an interesting model of ADP with dissimilar binding conformations at each NADH/ADP site in the GDH trimer, which explains the poorly understood strong binding but weak activation shown in experimental studies. In contrast, NADH showed similar inhibitory binding conformations at each NADH/ADP site. The structural analysis of the important residues in the NADH/ADP binding site presented in this paper may provide potential targets for mutation studies for allosteric drug design.