Engineering a mouse metallothionein on the cell surface of Ralstonia eutropha CH34 for immobilization of heavy metals in soil.

Here we describe targeting of the mouse metallothionein I (MT) protein to the cell surface of the heavy metal-tolerant Ralstonia eutropha (formerly Alcaligenes eutrophus) CH34 strain, which is adapted to thrive in soils highly polluted with metal ions. DNA sequences encoding MT were fused to the autotransporter beta-domain of the IgA protease of Neisseria gonorrhoeae, which targeted the hybrid protein toward the bacterial outer membrane. The translocation, surface display, and functionality of the chimeric MTbeta protein was initially demonstrated in Escherichia coli before the transfer of its encoding gene (mtb) to R. eutropha. The resulting bacterial strain, named R. eutropha MTB, was found to have an enhanced ability for immobilizing Cd2+ ions from the external media. Furthermore, the inoculation of Cd2+-polluted soil with R. eutropha MTB decreased significantly the toxic effects of the heavy metal on the growth of tobacco plants (Nicotiana bentamiana).

The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: observation of an enzyme-bound NAD-ketone adduct at 1.4 A resolution by X-ray crystallography.

Drosophila alcohol dehydrogenase (DADH) is an NAD+-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones. DADH is the member of the short-chain dehydrogenases/reductases family (SDR) for which the largest amount of biochemical data has been gathered during the last three decades. The crystal structures of one binary form (NAD+) and three ternary complexes with NAD+.acetone, NAD+.3-pentanone and NAD+.cyclohexanone were solved at 2.4, 2.2, 1. 4 and 1.6 A resolution, respectively. From the molecular interactions observed, the reaction mechanism could be inferred. The structure of DADH undergoes a conformational change in order to bind the coenzyme. Furthermore, upon binding of the ketone, a region that was disordered in the apo form (186-191) gets stabilized and closes the active site cavity by creating either a small helix (NAD+. acetone, NAD+.3-pentanone) or an ordered loop (NAD+.cyclohexanone). The active site pocket comprises a hydrophobic bifurcated cavity which explains why the enzyme is more efficient in oxidizing secondary aliphatic alcohols (preferably R form) than primary ones. Difference Fourier maps showed that the ketone inhibitor molecule has undergone a covalent reaction with the coenzyme in all three ternary complexes. Due to the presence of the positively charged ring of the coenzyme (NAD+) and the residue Lys155, the amino acid Tyr151 is in its deprotonated (tyrosinate) state at physiological pH. Tyr151 can subtract a proton from the enolic form of the ketone and catalyze a nucleophilic attack of the Calphaatom to the C4 position of the coenzyme creating an NAD-ketone adduct. The binding of these NAD-ketone adducts to DADH accounts for the inactivation of the enzyme. The catalytic reaction proceeds in a similar way, involving the same amino acids as in the formation of the NAD-ketone adduct. The p Kavalue of 9-9.5 obtained by kinetic measurements on apo DADH can be assigned to a protonated Tyr151 which is converted to an unprotonated tyrosinate (p Ka7.6) by the influence of the positively charged nicotinamide ring in the binary enzyme-NAD+form. pH independence during the release of NADH from the binary complex enzyme-NADH can be explained by either a lack of electrostatic interaction between the coenzyme and Tyr151 or an apparent p Kavalue for this residue higher than 10.0.

The refined crystal structure of Drosophila lebanonensis alcohol dehydrogenase at 1.9 A resolution.

Drosophila alcohol dehydrogenase (DADH; EC 1.1.1.1) is a NAD(H)-dependent oxidoreductase belonging to the short-chain dehydrogenases/reductases (SDR) family. This homodimeric enzyme catalyzes the dehydrogenation of alcohols to their respective ketones or aldehydes in the fruit-fly Drosophila, both for metabolic assimilation and detoxification purposes. The crystal structure of the apo form of DADH, one of the first biochemically characterized member of the SDR family, was solved at 1.9 A resolution by Patterson methods. The initial model was improved by crystallographic refinement accompanied by electron density averaging, R-factor=20.5%, R-free=23.8%.DADH subunits show an alpha/beta single domain structure with a characteristic NAD(H) binding motif (Rossmann fold). The peptide chain of a subunit is folded into a central eight-stranded beta-sheet flanked on each side by three alpha-helices. The dimers have local 2-fold symmetry. Dimer association is dominated by a four-helix bundle motif as well as two C-terminal loops from each subunit, which represent a unique structural feature in SDR enzymes with known structure. Three structural features are characteristic for the active site architecture. (1) A deep cavity which is covered by a flexible loop (33 residues) and the C-terminal tail (11 residues) from the neighboring subunit. The hydrophobic surface of the cavity is likely to increase the specificity of this enzyme towards secondary aliphatic alcohols. (2) The residues of the catalytic triad (Ser138, Tyr151, Lys155) are known to be involved in enzymatic catalysis in the first line. The Tyr151 OH group is involved in an ionic bond with the Lys155 side-chain. Preliminary electrostatic calculations have provided evidence that the active form of Tyr151 is a tyrosinate ion at physiological pH. (3) Three well-ordered water molecules in hydrogen bond distance to side-chains of the catalytic triad may be significant for the proton release steps in DADH catalysis.A ternary structure-based sequence alignment with ten members of the SDR family with known three-dimensional structure has suggested to define a model consisting of four groups of residues, which relates the observed low degree of sequence identity to quite similar folding patterns and nearly identical distributions of residues involved in catalysis.

Preliminary X-ray crystallographic studies on alcohol dehydrogenase from Drosophila.

The alcohol dehydrogenase (ADHase) enzyme catalyses the oxidation of alcohols to aldehydes or ketones using NAD+ as a cofactor. Functional ADHase from Drosophila lebanonensis is a dimer, with a monomeric molecular weight of 27,000 and with 254 residues in each polypeptide chain. Crystals of the protein have been grown with and without NAD+. Two crystal forms have been observed. Most crystals are plate-like, 0.05 mm in their shortest dimension and up to 0.4 mm in their longest dimension. These crystals are generally too small to diffract efficiently using conventional X-ray sources, so preliminary studies were carried out using the Synchrotron Radiation Source at the SERC Daresbury Laboratory. Twinning was a severe problem with this crystal form. The second form is grown in the absence of NAD+ but with DL-dithiothreitol present. These crystals grow more evenly and diffract to better than 2 A resolution. They are monoclinic, with cell dimensions, a = 81.24(6) A, b = 55.75(4) A, c = 109.60(7) A and beta = 94.26(9) degrees, space group P2(1). There are two dimers in the asymmetric unit, but at low resolution a rotated cell with one dimer per asymmetric unit can be obtained.

Sunflower metallothionein family characterisation. Study of the Zn(II)- and Cd(II)-binding abilities of the HaMT1 and HaMT2 isoforms.

Plant metallothioneins (MTs) constitute a family of small Cys-rich proteins capable of coordinating metal ions, significantly differing from microbial and animal MTs. They are divided into four subfamilies depending on the Cys pattern in their sequence. In this work, the MT system of the sunflower plant (Helianthus annuus) has been defined, with ten genes coding for MTs (HaMT) belonging to the four plant MT subfamilies; three HaMT1, four HaMT2, one HaMT3 and two HaMT4 isoforms. The gene expression pattern and capacity to confer metal resistance to yeast cells have been analysed for at least one member of each subfamily. The divalent metal ion-binding abilities of HaMT1-2 and HaMT2-1 (the isoforms encoded by the most abundantly expressed HaMT1 and HaMT2 isogenes) have been characterised, as HaMT3 and HaMT4 were previously studied. Those isoforms constitute an optimum material to study the effect of Cys number variability on their coordination abilities, as they exhibit additional Cys residues regarding the canonical Cys pattern of each subfamily. Our results show that the variation in the number of Cys does not drastically modify their M(II)-binding abilities, but instead modulates the degree of heterogeneity of the corresponding recombinant syntheses. Significantly, the Zn(II)-HaMT1 complexes were highly susceptible to proteolytic cleavage. The recombinant Cd-MT preparations of both isoforms exhibit significant acid-labile sulphide content-Cd6S8 or Cd7S7 species. Overall results suggest that HaMT2-1 is probably associated with Cd(II) detoxification, in contrast to HaMT1-2, which may be more related to physiological functions, such as metal ion transport and delivery.

Synthesis of Drosophila melanogaster alcohol dehydrogenase in yeast.

Expression systems for the heterologous expression of Drosophila melanogaster alcohol dehydrogenase (ADH) in Saccharomyces cerevisiae have been designed, analyzed and compared. Four different yeast/Escherichia coli shuttle vectors were constructed and used to transform four different yeast strains. Expression was detectable in ADH- yeast strains, from either a constitutive promoter, yeast ADH1 promoter (ADCp), or a regulated promoter, yeast GALp. The highest amount of D. melanogaster ADH was obtained from a multicopy plasmid with the D. melanogaster Adh gene expressed constitutively under the control of yeast ADCp promoter. The D. melanogaster enzyme was produced in cell extracts, as assessed by Coomassie blue staining and Western blotting after polyacrylamide-gel electrophoresis and it was fully active and able to complement the yeast ADH deficiency. Results show that D. melanogaster ADH subunits synthesized in yeast are able to assemble into functional dimeric forms. The synthesized D. melanogaster ADH represents up to 3.5% of the total extracted yeast protein.

Protein engineering of Drosophila alcohol dehydrogenase. The hydroxyl group of Tyr152 is involved in the active site of the enzyme.

Drosophila alcohol dehydrogenase is the most studied member of the family of short-chain alcohol dehydrogenases, although its tridimensional structure still remains unknown. We have engineered a Drosophila alcohol dehydrogenase in which tyrosine-152, an invariant residue in all members of the family, has been substituted by phenylalanine. The mutated gene has been expressed in yeast and pure mutant enzyme has been prepared by a one-step FPLC chromatographic procedure. Drosophila alcohol dehydrogenase-phenylalanine-152 shows no enzymatic activity. This result suggests not only that tyrosine-152 could constitute an essential building block of the active site but also that its hydroxyl group is directly involved in the redox reaction catalyzed by the enzyme.

Drosophila lebanonensis ADH: analysis of recombinant wild-type enzyme and site-directed mutants. The effect of restoring the consensus sequence in two positions.

Unique amino acid substitutions occur in D. lebanonensis ADH. They are found within the putative NAD(+)-binding domain and affect residues that are otherwise highly conserved in all other species of the genus. To restore the consensus amino acids, we have constructed an expression system for this enzyme in E. coli, and engineered two mutants, Ala13Gly and Asn56Thr. The biochemical and kinetic features of these retromutants are consistent with increased catalytic efficiency and thermal stability. Thus, results show that wild-type D. lebanonensis ADH can be improved by site-directed mutagenesis.

Identification of reactive tyrosine residues in cysteine-reactive dehydrogenases. Differences between liver sorbitol, liver alcohol and Drosophila alcohol dehydrogenases.

Modification of tyrosine residues with tetranitromethane and reversible sulphite protection of cysteine residues were tested on three dehydrogenases of two families. In liver alcohol dehydrogenase no Tyr residue is appreciably labelled, while in the homologous sorbitol dehydrogenase Tyr-109 is specifically labelled; the difference corresponds to a segment correlating with subunit interactions and the different quaternary structures of the proteins. In Drosophila alcohol dehydrogenase, Tyr modification is multiple, and the results show the presence of two different states of Cys residues, reactive in the presence and absence of cupric ions, respectively. Super-activation with cyanide was also noticed after S-sulphocysteine protection. The results demonstrate the possibility of identification of specific Tyr residues in proteins with reversibly protected Cys residues.

Drosophila alcohol dehydrogenase: evaluation of Ser139 site-directed mutants.

Drosophila alcohol dehydrogenase (DADH) belongs to the large and highly heterogeneous (15-30% residue identity) short-chain dehydrogenase/reductase family (SDR). It is the only reported member that oxidizes mainly ethanol and 2-propanol among other alcohols. To confirm the role of Ser139 we constructed two site-directed mutants, Ser139Ala and Ser139Cys, which show no enzymatic activity. Molecular replacement and data from crystallographically refined 3D structures confirm the position of Ser139, whose hydroxyl group faces the cleft of the presumed catalytic pocket, very close to Tyr152 and Lys156. Thus, consistent with the constitution of the catalytic triad of other SDR, our results suggest that Ser139 of DADH is directly involved in the catalytic reaction.

Effect of site-directed mutagenesis on conserved positions of Drosophila alcohol dehydrogenase.

Tyr152 and Lys156 may be functionally important residues in Drosophila ADH as they are conserved in the genus and in all short-chain dehydrogenases. In addition, unaltered Gly positions could have a crucial role in the building of the structural framework. We have modified Drosophila ADH and expressed the mutant forms in E. coli. Mutation of Tyr152 to Glu or Gln, Lys156 to Ile, Gly184 to Leu, and the double mutant Gly130 to Cys and Gly133 to Ile, all rendered, with different substrates and at different pHs, an inactive enzyme. Results suggest that Tyr152 and Lys156 are involved in catalysis and that Gly130, Gly133 and Gly184 contribute substantially to the structure of the active form.

MTO: the second member of a Drosophila dual copper-thionein system.

Drosophila MTO metal binding features were analyzed for comparison with MTN, the paralogous Drosophila metallothionein, and to classify MTO as either zinc- or copper-thionein. This was achieved by a combination of in vivo, in vitro and in silico methodologies. All the results unambiguously classified MTO as a second Drosophila copper-thionein, putting Drosophila forward as the only metazoan in which any zinc-thionein has still to be reported. Interestingly, experimental data only showed minor differences in the coordinative behavior of both MTs, but provided a characteristic spectroscopic fingerprint, revealing the possible binding of chloride anions in certain metal-MTO aggregates.

Drosophila MTN: a metazoan copper-thionein related to fungal forms.

Two Drosophila metallothioneins (MT) have been reported: MTN, a 40 residue peptide including 10 Cys, and MTO, a 43 residue peptide including 12 Cys. However, neither functional nor evolutionary analyses for either of the Drosophila MT are available. Here, heterologous expression of Mtn in Escherichia coli is reported. The metal binding abilities of the Cu- and Zn-MTN complexes conformed in vivo, as well as the features of the Cd- and Cu-aggregates produced by metal replacement in vitro, have been determined by atomic emission spectrometry, circular dichroism and electrospray ionization mass spectrometry. Primary structure relationships with other MT have been examined. The results indicate a close resemblance of MTN to fungal copper-thioneins.

Bioaccumulation of heavy metals with protein fusions of metallothionein to bacterial OMPs.

In view of potential biotechnological applications, eukaryotic metallothioneins (MTs) have been expressed in Escherichia coli as fusions to membrane or membrane-associated proteins such as LamB, the peptidoglycan-associated lipoprotein protein (PAL) or a hybrid Lpp/OmpA carrier sequence. The use of different anchors enables the MT moiety to be targeted into various cell compartments thus bringing the metal-binding ability of the resulting hybrids to specific sites of the cell structure. To this end, both full-size and partial sequences of the human or mouse MTs have been genetically fused to: i) the permissive site 153 of the LamB sequence, which loops out the MT to the external medium; ii) the N-terminus of a PAL variant devoid of its N-terminal cystein, which targets expression of the fusion into the periplasm; and iii) the C-terminus of Lpp-OmpA, for anchoring the MT to the outer membrane protein as an N-terminal fusion. Each type of fusion presented a distinct behavior in terms of expression, stability and ability to endow E. coli cells an enhanced accumulation of Cd2+, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions. The expression in vivo of metalloproteins bound to bacterial envelope structures opens a way to design biomass with specific metal-binding properties.

Engineering outer-membrane proteins in Pseudomonas putida for enhanced heavy-metal bioadsorption.

Metallothioneins (MTs) are small, cysteine-rich proteins with a strong metal-binding capacity that are ubiquitous in the animal kingdom. Recombinant expression of MT fused to outer-membrane components of gram-negative bacteria may provide new methods to treat heavy-metal pollution in industrial sewage. In this work, we have engineered Pseudomonas putida, a per se highly robust microorganism able to grow in highly contaminated habitats in order to further increase its metal-chelating ability. We report the expression of a hybrid protein between mouse MT and the beta domain of the IgA protease of Neisseria in the outer membrane of Pseudomonas cells. The metal-binding capacity of such cells was increased three-fold. The autotranslocating capacity of the beta domain of the IgA protease of Neisseria, as well as the correct anchoring of the transported protein into the outer membrane, have been demonstrated for the first time in a member of the Pseudomonas genus.

A new insight into the Ag+ and Cu+ binding sites in the metallothionein beta domain.

The copper(I) and silver(I) binding properties of the beta fragment of recombinant mouse metallothionein I have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn3-beta MT at pH 7 or apo-beta MT at pH 3 with Cu+ leads to the formation of species having the same stoichiometry and structure: Cu6-beta MT, Cu7-beta MT and Cu10-beta MT. In the first stage of the titration of Zn3-beta MT with Cu+ at pH 7 one additional species of formula Cu4Zn1-beta MT was detected. In contrast, the titration of Zn3-beta MT at pH 7.5 and of apo-beta MT at pH 2.5 with Ag+ proceeds through different reaction pathways, affording ZnxAg3-beta MT, Ag6-beta MT and Ag9-beta MT or Ag3-beta MT, Ag6-beta MT and Ag9-beta MT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag6-beta MT and Ag9-beta MT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned.

Binding of excess cadmium(II) to Cd7-metallothionein from recombinant mouse Zn7-metallothionein 1. UV-VIS absorption and circular dichroism studies and theoretical location approach by surface accessibility analysis.

A mouse metallotbionein (MT) 1 expression system has been constructed that renders recombinant MT as a high purity Zn-coordinated protein. Spectral changes in absorption and circular dichroism following the addition of up to 7 mol equivalents of Cd2+ to recombinant Zn7-MT showed that it behaves like the native protein. Exposure of Cd7-MT to Cd2+ resulted in further binding of these ions to the protein, although saturation was not achieved on the addition of up to 22 mol equivalents of Cd2+ to Zn7-MT. Spectral data are compatible with a model in which the first four additional Cd2+ ions are bound to Cd7-MT via sulfur atoms, and indicate that no further thiol groups are involved in the binding of the excess Cd(II) over 11. Cd2+ ions bound in excess to Cd7-MT appear to have lower binding constants as exposure of Cdn-MT (n > 7) species to Cbelex-100 retrieved Cd7-MT. Based on the X-ray data, the accessible surface areas of sulfur atoms in Cd5,Zn2-MT 2 were calculated. This led us to propose that the coordination of the first three additional Cd(II) ions to Cd7-MT proceeds by means of S-Met1-O-Met1, S-Cys7-S-Cys13 and S-Cys5-S-Cys26 pairs. Finally, comparison of the behavior of the entire MT with that of the recombinant alpha MT and beta MT subunits indicates that mutual influences may not be negligible.

Genesis of Drosophila ADH: the shaping of the enzymatic activity from a SDR ancestor.

Drosophila alcohol dehydrogenase (ADH) is an NAD(H)-dependent oxidoreductase that catalyzes the oxidation of alcohols and aldehydes. Structurally and biochemically distinct from all the reported ADHs (typically, the mammalian medium-chain dehydrogenase/reductase-ethanol-metabolizing enzyme), it stands as the only small-alcohol transforming system that has originated from a short-chain dehydrogenase/reductase (SDR) ancestor. The crystal structures of the apo, binary (E.NAD(+)) and three ternary (E.NAD(+).acetone, E.NAD(+).3-pentanone and E.NAD(+).cyclohexanone) forms of Drosophila lebanonensis ADH have allowed us to infer the structural and kinetic features accounting for the generation of the ADH activity within the SDR lineage.

The response of the different soybean metallothionein isoforms to cadmium intoxication.

Cadmium is a highly toxic heavy metal for both plants and animals. The presence of Cd in agricultural soils is of major concern regarding its entry into the food chain, since Cd compounds are readily taken up by plants, and accumulated in edible parts due to their high solubility. In this study, we first demonstrate the high capacity for Cd concentration of soybean grains. Consequently, we considered the study and characterization of the molecular determinants of Cd accumulation -such as metallothioneins (MT)- to be of major practical importance. We report here the first characterization of the soybean MT system, with the identification of nine genes (one of which is a truncated pseudogene), belonging to the four plant MT types. The most highly expressed of each type was chosen for further function analysis. All of them are expressed at high levels in soybean tissues: GmMT1, GmMT2 and GmMT3 in roots, shoots and seeds, and GmMT4 only in seeds. The corresponding recombinant soybean MTs, synthesized in Escherichia coli cells cultured in metal supplemented media, exhibit greater cadmium than zinc binding capacity. These results suggest a definite role of GmMTs in Cd(II) accumulation as one of the main responses of soybean to an overload of this metal.

Purification and molecular characterization of alcohol dehydrogenase from Drosophila hydei: conservation in the biochemical features of the enzyme in several species of Drosophila.

Alcohol dehydrogenase (ADH) has been purified from Drosophila hydei. Biochemical investigations show that the native enzyme is a dimer consisting of two identical subunits with molecular weight 27,000. The pH optimum values of pure enzyme preparations are 7.9 and 9.4. The pI values are 8.83 and 8.41. Substrate specificities have been characterized. Km(app) values are lowest for propan-2-ol and butan-2-ol and Vmax(app) values are highest for these two substrates. The amino acid composition has been determined. Peptide mapping experiments performed after trypsin digestion of the enzyme allow the identification of 24 peptides. Peptides comprising 64% of the amino acid residues have also been purified by high-performance liquid chromatography (HPLC), and their N-terminal residues and amino acid composition determined. Results are compared with the amino acid sequence of ADH from D. melanogaster Adhs [Thatcher, D. R. (1980). Biochem. J. 187:875]. When data on the biochemical and structural characterization of ADH from D. hydei are compared with data from other species of Drosophila, clear homologies are observed.