Investigation of the nature of the two metal-binding sites in 5-aminolaevulinic acid dehydratase from Escherichia coli.
Ontology highlight
ABSTRACT:
SUBMITTER: Spencer P
PROVIDER: S-EPMC1138172 | biostudies-other | 1994 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Experiments are described in which the individual properties of the two 5-aminolaevulinic acid (ALA) binding sites, the A-site and the P-site, of 5-aminolaevulinic acid dehydratase (ALAD) have been investigated. The ALA binding affinity at the A-site is greatly enhanced (at least 10-fold) on the binding of the catalytic metal ion (bound at the alpha-site). The nature of the catalytic metal ion, Mg2+ or Zn2+, also gave major variations in the substrate Km, P-site affinity for ALA, the effect of potassium and phosphate ions and the pH-dependence of substrate binding. Modification of the P-site by reaction of the enzyme-substrate Schiff base with NaBH4 and analysis of the reduced adduct by electro-spray mass spectrometry indicated a maximum of 1 mol of substrate incorporated/mol of subunit, correlating with a linear loss of enzyme activity. The reduced Schiff-base adduct was used to investigate substrate binding at the A-site by using rate-of-dialysis analysis. The affinity for ALA at the A-site of Mg alpha Zn beta ALAD was found to determine the Km for the reaction and was pH-dependent, with its affinity increasing from 1 mM at pH 6 to 70 microM at pH 8.5. The affinity of ALA at the P-site of Zn alpha An beta ALAD is proposed to limit the Km at pH values above 7, since the measured Kd for ALA at the A-site in 45 microM Tris, pH 8, was well below the observed Km (600 microM) under the same conditions. The amino group of the ALA molecule bound at the P-site was identified as a critical binding component for the A-site, explaining why ALA binding to ALAD is ordered, with the P-site ALA binding first. Structural requirements for ALA binding at the A- and P-sites have been identified: the P-site requires the carbonyl and carboxylate groups, whereas the A-site requires the amino, carbonyl and carboxylate groups of the substrate.
| S-EPMC1136443 | biostudies-other
Project description:5-Aminolaevulinic acid dehydratase (ALAD) from a recombinant strain of Escherichia coli was purified to homogeneity. The enzyme is a homo-octamer of subunit M(r) 36554 +/- 17. Enzyme activity was dependent on the presence of Zn2+ ions and an exogenous thiol. Two molar equivalents of Zn2+ are bound/mol of subunit under reducing conditions. On exposure to the metal chelator EDTA, the two Zn2+ ions are removed, giving an inactive metal-depleted apo-ALAD. On oxidation of holo-ALAD, two disulphide bonds are formed with the loss of 1 mol of Zn2+/mol of subunit. The formation of the first disulphide led to the loss of catalytic activity. Replacement of the two bound Zn2+ ions with Co2+ resulted in the formation of a green protein with a spectrum indicative of the presence of charge-transfer bands from one or more cysteine-Co2+ ligands. While Mg2+ could not activate apo-ALAD alone, it was able to substitute for the second molar equivalent of bound Zn2+, leading to a further 4-fold stimulation in activity. The four cysteine residues involved in the formation of the two disulphide bonds were identified by protein-chemistry studies and were all located in a region of the protein extending from amino acid residues 120-134. Protein sequence data obtained in the present study has permitted the resolution of several differences between the published gene-derived protein sequences for ALAD from E. coli.
| S-EPMC1132412 | biostudies-other
Project description:A comparative genomic analysis predicted that many members of the under-characterized COG0523 subfamily of putative P-loop GTPases function in metal metabolism. In this work we focused on the uncharacterized Escherichia coli protein YeiR by studying both the physiology of a yeiR mutant and the in vitro biochemical properties of YeiR expressed as a fusion with the maltose-binding protein (YeiR-MBP). Our results demonstrate that deletion of yeiR increases the sensitivity of E. coli to EDTA or cadmium, and this phenotype is linked to zinc depletion. In vitro, the tagged protein binds several Zn(2+) ions with nanomolar affinity and oligomerizes in the presence of metal. The GTPase activity of YeiR is similar to that measured for other members of the group, but GTP hydrolysis is enhanced by Zn(2+) binding. These results support the predicted connection between the COG0523 P-loop GTPases and roles in metal homeostasis.
| S-EPMC3574555 | biostudies-literature
Project description:D-xylonate dehydratase YjhG from Escherichia coli can convert D-xylonate into 2-keto-3-deoxy- D-xylonate (KDX), and is a key enzyme in the biosynthesis of 1,2,4-butanetriol and other chemicals. However, the biochemical properties of YjhG still remain unknown. In this study, the activity assay method for YjhG was established based on semicarbazide method, in which KDX reacts with semicarbazide reagent, and is further quantified by high-resolution mass spectrometry. The effect of reaction conditions on YjhG activity was determined in vitro using purified His-tagged YjhG protein. This enzyme showed maximal activity at 30°C and pH 8.0. Bivalent metal ions such as Mg(2+) and Mn(2+) activated, whereas Ni(2+) and Zn(2+) inhibited the activity of YjhG. Under optimal conditions, the Km and Vmax values were 4.88 mM and 78.62 ?M l(-1)h(-1), respectively, when using D-xylonate as a substrate. Amino acids sequence alignments and catalytic properties analysis revealed that YjhG might be a member of IlvD/EDD family. Results obtained in this study may lay a foundation for further investigation on YjhG and will benefit its application in biosynthesis of related chemicals.
| S-EPMC4601382 | biostudies-literature
Project description:D-Serine dehydratase from Escherichia coli is a member of the β-family (fold-type II) of the pyridoxal 5'-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97Å-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55Å resolution. The structure of DSD reveals a larger pyridoxal 5'-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the β-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.
| S-EPMC7380123 | biostudies-literature
Project description:Metal binding by members of the growth hormone (GH) family of hematopoietic cytokines has been a subject of considerable interest. However, beyond appreciation of its role in reversible packing of GH proteins in secretory granules, the molecular mechanisms of metal binding and granule formation remain poorly understood. Here, we investigate metal binding by a GH family member prolactin (PRL) using paramagnetic metal titration and chelation experiments. Cu2+-mediated paramagnetic relaxation enhancement measurements identified two partial metal-binding sites on the opposite faces of PRL composed of residues H30/H180 and E93/H97, respectively. Coordination of metal ions by these two sites causes formation of inter-molecular bridges between the PRL protomers and enables formation of reversible higher aggregates. These findings in vitro suggest a model for reversible packaging of PRL in secretory granules. The proposed mechanism of metal-promoted PRL aggregation lends insight and support to the previously suggested role of metal coordination in secretory granule formation by GH proteins.
| S-EPMC9034190 | biostudies-literature
Project description:In Escherichia coli and other bacteria, nickel uptake is regulated by the transcription factor NikR. Nickel binding at high-affinity sites in E. coli NikR (EcNikR) facilitates EcNikR binding to the nik operon, where it then suppresses transcription of genes encoding the nickel uptake transporter, NikABCDE. A structure of the EcNikR-DNA complex suggests that a second metal-binding site is also present when NikR binds to the nik operon. Moreover, this co-crystal structure raises the question of what metal occupies the second site under physiological conditions: K(+), which is present in the crystal structure, or Ni(2+), which has been proposed to bind to low- as well as high-affinity sites on EcNikR. To determine which ion is preferred at the second metal-binding site and the physical basis for any preference of one ion over another in both the second metal-binding site and the high-affinity sites, we conducted a series of detailed molecular simulations on the EcNikR structure. Simulations that place Ni(2+) at high-affinity sites lead to stable trajectories with realistic ion-ligand distances and geometries, while simulations that place K(+) at these sites lead to conformational changes in the protein that are likely unfavorable for ion binding. By contrast, simulations on the second metal site in the EcNikR-DNA complex lead to stable trajectories with realistic geometries regardless of whether K(+) or Ni(2+) occupies this site. Electrostatic binding free energy calculations, however, suggest that EcNikR binding to DNA is more favorable when the second metal-binding site contains K(+). An analysis of the energetic contributions to the electrostatic binding free energy suggests that, while the interaction between EcNikR and DNA is more favorable when the second site contains Ni(2+), the large desolvation penalty associated with moving Ni(2+) from solution to the relatively buried second site offsets this favorable interaction term. Additional free energy simulations that account for both electrostatic and non-electrostatic effects argue that EcNikR binding to DNA is most favorable when the second site contains a monovalent ion the size of K(+). Taken together, these data suggest that the EcNikR structure is most stable when Ni(2+) occupies high-affinity sites and that EcNikR binding to DNA is more favorable when the second site contains K(+).
| S-EPMC3579654 | biostudies-literature
Project description:Most organisms use glutathione to regulate intracellular thiol redox balance and protect against oxidative stress; protozoa, however, utilize trypanothione for this purpose. Trypanothione biosynthesis requires ATP-dependent conjugation of glutathione (GSH) to the two terminal amino groups of spermidine by glutathionylspermidine synthetase (GspS) and trypanothione synthetase (TryS), which are considered as drug targets. GspS catalyzes the penultimate step of the biosynthesis-amide bond formation between spermidine and the glycine carboxylate of GSH. We report herein five crystal structures of Escherichia coli GspS in complex with substrate, product or inhibitor. The C-terminal of GspS belongs to the ATP-grasp superfamily with a similar fold to the human glutathione synthetase. GSH is likely phosphorylated at one of two GSH-binding sites to form an acylphosphate intermediate that then translocates to the other site for subsequent nucleophilic addition of spermidine. We also identify essential amino acids involved in the catalysis. Our results constitute the first structural information on the biochemical features of parasite homologs (including TryS) that underlie their broad specificity for polyamines.
| S-EPMC1698887 | biostudies-literature
Project description:A computational search was carried out to identify additional targets for the Escherichia coli OxyR transcription factor. This approach predicted OxyR binding sites upstream of dsbG, encoding a periplasmic disulfide bond chaperone-isomerase; upstream of fhuF, encoding a protein required for iron uptake; and within yfdI. DNase I footprinting assays confirmed that oxidized OxyR bound to the predicted site centered 54 bp upstream of the dsbG gene and 238 bp upstream of a known OxyR binding site in the promoter region of the divergently transcribed ahpC gene. Although the new binding site was near dsbG, Northern blotting and primer extension assays showed that OxyR binding to the dsbG-proximal site led to the induction of a second ahpCF transcript, while OxyR binding to the ahpCF-proximal site leads to the induction of both dsbG and ahpC transcripts. Oxidized OxyR binding to the predicted site centered 40 bp upstream of the fhuF gene was confirmed by DNase I footprinting, but these assays further revealed a second higher-affinity site in the fhuF promoter. Interestingly, the two OxyR sites in the fhuF promoter overlapped with two regions bound by the Fur repressor. Expression analysis revealed that fhuF was repressed by hydrogen peroxide in an OxyR-dependent manner. Finally, DNase I footprinting experiments showed OxyR binding to the site predicted to be within the coding sequence of yfdI. These results demonstrate the versatile modes of regulation by OxyR and illustrate the need to learn more about the ensembles of binding sites and transcripts in the E. coli genome.
| S-EPMC95352 | biostudies-literature
Project description:GlxI (glyoxalase I) isomerizes the hemithioacetal formed between glutathione and methylglyoxal. Unlike other GlxI enzymes, Escherichia coli GlxI exhibits no activity with Zn(2+) but maximal activation with Ni(2+). To elucidate further the metal site in E. coli GlxI, several approaches were undertaken. Kinetic studies indicate that the catalytic metal ion affects the k (cat) without significantly affecting the K (m) for the substrate. Inductively coupled plasma analysis and isothermal titration calorimetry confirmed one metal ion bound to the enzyme, including Zn(2+), which produces an inactive enzyme. Isothermal titration calorimetry was utilized to determine the relative binding affinity of GlxI for various bivalent metals. Each metal ion examined bound very tightly to GlxI with an association constant ( K (a))>10(7) M(-1), with the exception of Mn(2+) ( K (a) of the order of 10(6) M(-1)). One of the ligands to the catalytic metal, His(5), was altered to glutamine, a side chain found in the Zn(2+)-active Homo sapiens GlxI. The affinity of the mutant protein for all bivalent metals was drastically decreased. However, low levels of activity were now observed for Zn(2+)-bound GlxI. Although this residue has a marked effect on metal binding and activation, it is not the sole factor determining the differential metal activation between the human and E. coli GlxI enzymes.
| S-EPMC1223881 | biostudies-other