Covalent inhibition of P. falciparum ferredoxin-NADP+ reductase: Exploring alternative strategies for the development of new antimalarial drugs.

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads.

Analysis of Cysteine Post Translational Modifications Using Organic Mercury Resin.

The wide reactivity of the thiol group enables the formation of a variety of reversible, covalent modifications on cysteine residues. S-nitrosylation, like many other post-translational modifications, is site selective, reversible, and necessary for a wide variety of fundamental cellular processes. The overall abundance of S-nitrosylated proteins and reactivity of the nitrosyl group necessitates an enrichment strategy for accurate detection with adequate depth. Herein, a method is presented for the enrichment and detection of endogenous protein S-nitrosylation from complex mixtures of cell or tissue lysate utilizing organomercury resin. Minimal adaptations to the method also support the detection of either S-glutathionylation or S-acylation using the same enrichment platform. When coupled with high accuracy mass spectrometry, these methods enable a site-specific level of analysis, facilitating the curation comparable datasets of three separate cysteine post-translational modifications. © 2018 by John Wiley & Sons, Inc.

Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

Ruthenium dihydroxybipyridine complexes are tumor activated prodrugs due to low pH and blue light induced ligand release.

Ruthenium drugs are potent anti-cancer agents, but inducing drug selectivity and enhancing their modest activity remain challenging. Slow Ru ligand loss limits the formation of free sites and subsequent binding to DNA base pairs. Herein, we designed a ligand that rapidly dissociates upon irradiation at low pH. Activation at low pH can lead to cancer selectivity, since many cancer cells have higher metabolism (and thus lower pH) than non-cancerous cells. We have used the pH sensitive ligand, 6,6'-dihydroxy-2,2'-bipyridine (66'bpy(OH)2), to generate [Ru(bpy)2(66'(bpy(OH)2)](2+), which contains two acidic hydroxyl groups with pKa1=5.26 and pKa2=7.27. Irradiation when protonated leads to photo-dissociation of the 66'bpy(OH)2 ligand. An in-depth study of the structural and electronic properties of the complex was carried out using X-ray crystallography, electrochemistry, UV/visible spectroscopy, and computational techniques. Notably, RuN bond lengths in the 66'bpy(OH)2 complex are longer (by ~0.3Å) than in polypyridyl complexes that lack 6 and 6' substitution. Thus, the longer bond length predisposes the complex for photo-dissociation and leads to the anti-cancer activity. When the complex is deprotonated, the 66'bpy(O(-))2 ligand molecular orbitals mix heavily with the ruthenium orbitals, making new mixed metal-ligand orbitals that lead to a higher bond order. We investigated the anti-cancer activities of [Ru(bpy)2(66'(bpy(OH)2)](2+), [Ru(bpy)2(44'(bpy(OH)2)](2+), and [Ru(bpy)3](2+) (44'(bpy(OH)2=4,4'-dihydroxy-2,2'-bipyridine) in HeLa cells, which have a relatively low pH. It is found that [Ru(bpy)2(66'(bpy(OH)2)](2+) is more cytotoxic than the other ruthenium complexes studied. Thus, we have identified a pH sensitive ruthenium scaffold that can be exploited for photo-induced anti-cancer activity.

Speciation analysis of mercury in water samples by dispersive liquid-liquid microextraction coupled to capillary electrophoresis.

In this study, a method of pretreatment and speciation analysis of mercury by dispersive liquid-liquid microextraction along with CE was developed. The method was based on the fact that mercury species including methylmercury (MeHg), ethylmercury (EtHg), phenylmercury (PhHg), and Hg(II) were complexed with 1-(2-pyridylazo)-2-naphthol to form hydrophobic chelates and l-cysteine could displace 1-(2-pyridylazo)-2-naphthol to form hydrophilic chelates with the four mercury species. Factors affecting complex formation and extraction efficiency, such as pH value, type, and volume of extractive solvent and disperser solvent, concentration of the chelating agent, ultrasonic time, and buffer solution were investigated. Under the optimal conditions, the enrichment factors were 102, 118, 547, and 46, and the LODs were 1.79, 1.62, 0.23, and 1.50 µg/L for MeHg, EtHg, PhHg, and Hg(II), respectively. Method precisions (RSD, n = 5) were in the range of 0.29-0.54% for migration time, and 3.08-7.80% for peak area. Satisfactory recoveries ranging from 82.38 to 98.76% were obtained with seawater, lake, and tap water samples spiked at three concentration levels, respectively, with RSD (n = 5) of 1.98-7.18%. This method was demonstrated to be simple, convenient, rapid, cost-effective, and environmentally benign, and could be used as an ideal alternative to existing methods for analyzing trace residues of mercury species in water samples.

Copper(II) complex formation processes of alloferon I with point mutation H1K; combined spectroscopic and potentiometric studies.

Mononuclear and polynuclear complexes of the alloferon I with point mutation (H1K) Lys-Gly-Val-Ser-Gly-His(6)-Gly-Gln-His(9)-Gly-Val-His(12)-Gly (AlloK) and its acetylated derivative Ac-Lys-Gly-Val-Ser-Gly-His(6)-Gly-Gln-His(9)-Gly-Val-His(12)-Gly (Ac-AlloK) have been studied by potentiometric, UV-visible, CD, EPR spectroscopic and mass spectrometry (MS) methods. The high water solubility of the resulting metal complexes allowed us to obtain a complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 4:1 for AlloK while to 3:1 for Ac-AlloK. At physiological pH 7.4 and the metal-to-ligand 1:1molar ratio the AlloK peptide forms the CuL complex with the 4N {NH(2), N(-), 2N(Im)} binding mode. In the Cu(II)-AlloK 4:1 system in wide pH 6.5-10 range the Cu(4)H(-7)L complex dominates with the 3N {NH(2),2N(-)} 3×{N(Im),2N(-)} coordination mode. Imidazole nitrogen donor atoms are the primary and exclusive metal binding sites of Ac-AlloK. For Ac-AlloK and 1:1 metal-to-ligand molar ratio the CuHL complex with the 3N {3N(Im)} binding sites in pH 4.5-7.5 range is present in solution. The amine nitrogen donor and all of the histidine residues can be considered to be independent metal-binding sites in the species formed in the systems studied. As a consequence, tri- (for the Ac-AlloK) and tetra-nuclear (for the AlloK peptide) complexes for the metal-to-ligand 3:1 and 4:1molar ratios, respectively, are present in the solution.

Investigation of the reactivity between a ruthenium hexacationic prism and biological ligands.

The relative affinity of the cationic triangular metallaprism, [(pCH(3)C(6)H(4)Pr(i))(6)Ru(6)(tpt)(2)(dhbq)(3)](6+) ([1](6+)), for various amino acids, ascorbic acid, and glutathione (GSH) has been studied at 37 °C in aqueous solutions at pD 7, using NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS). The metallaprism [1](6+), which is constituted of six (pCH(3)C(6)H(4)Pr(i))Ru corners bridged by three 1,4-benzoquinonato (dhbq) ligands and connected by two 2,4,6tri(pyridin4yl)1,3,5-triazine (tpt) triangular panels, disassembled in the presence of Arg, His, and Lys, while it remains intact with Met. Coordination to the imidazole nitrogen atom in His or to the basic NH/NH(2) groups in Arg and Lys displaces the dhbq and tpt ligands from the (p-cymene)Ru units, and subsequent coordination to the amino and carboxylato groups forms stable N,N,O metallacycles. The binding to amino acids proceeds rapidly, as determined by NMR spectroscopy. Interestingly, solutions of [1](6+) are able to catalyze oxidation of the thiol group of Cys and GSH to give the corresponding disulfides and of ascorbic acid to give the corresponding dehydroascorbic acid. Competition experiments with Arg, Cys, His, and Lys show the simultaneous formation of one single adduct, the (p-cymene)Ru-His complex, and oxidation of Cys to cystine. Furthermore, the (p-cymene)Ru-His complex formed upon the addition of His to [1][CF(3)SO(3)](6) is able to oxidize Cys to cystine much more efficiently than [1](6+). These results provide evidence against interaction with proteins as process in the release of encapsulated guest molecules. Oxidation of Cys and GSH to give the corresponding disulfides may explain the in vitro anticancer activity of [1](6+).

Reversible fluorescent probe for highly selective and sensitive detection of mercapto biomolecules.

A coumarin-derived complex, Hg(2)L(2), was reported as a highly sensitive and selective probe for the detection of mercapto biomolecules in aqueous solution. The addition of Cys to a 99% aqueous solution of Hg(2)L(2) resulted in rapid and remarkable fluorescence OFF-ON (emission at 525 nm) due to the ligand-exchange reaction of Cys with L coordinated to Hg(2+). The increased fluorescence can be completely quenched by Hg(2+) and recovered again by the subsequent addition of Cys. Such a fluorescence OFF-ON circle can be repeated at least 10 times by the alterative addition of Cys and Hg(2+) to the solution of Hg(2)L(2), indicating that it can be used as a convertible and reversible probe for the detection of Cys. The interconversion of Hg(2)L(2) and L via the decomplexation/complexation by the modulation of Cys/Hg(2+) was definitely verified from their crystal structures. Other competitive amino acids without a thiol group cannot induce any fluorescence changes, implying that Hg(2)L(2) can selectively determine mercapto biomolecules. Using confocal fluorescence imaging, L/Hg(2)L(2) as a pair of reversible probes can be further applied to track and monitor the self-detoxification process of Hg(2+) ions in SYS5 cells.

A mechanistic approach for the DNA binding of chiral enantiomeric L- and D-tryptophan-derived metal complexes of 1,2-DACH: cleavage and antitumor activity.

A new chiral series of potential antitumor metal-based complexes 1-3(a and b) of L- and D-tryptophan have been synthesized and thoroughly characterized. Both enantiomers of 1-3 bind DNA noncovalently via phosphate interaction with slight preference of metal center for covalent coordination to nucleobases. The K(b) values of L-enantiomer, however, possess higher propensity for DNA binding in comparison with the D-enantiomeric analogs. The relative trend in K(b) values is as follows: 2(a) > 2(b) > 3(a) > 1(a) > 3(b) > 1(b). These observations together with the findings of circular dichoric and fluorescence studies reveal maximal potential of L-enantiomeric form of copper complex to bind DNA, thereby exerting its therapeutic effect. The complex 2a exhibits a remarkable DNA cleavage activity with pBR322DNA in the presence of different activators such as H(2) O(2) , ascorbic acid, 3-mercaptopropionic acid, and glutathione, suggesting the involvement of active oxygen species for the DNA scission. In vitro anticancer activity of complexes 1-3(a) were screened against 14 different human carcinoma cell lines of different histological origin, and the results reveal that 2a shows significant antitumor activity in comparison with both 1a and 3a and is particularly selective for MIAPACA2 (pancreatic cancer cell line).

Dynamic labeling strategy with 204Hg-isotopic methylmercurithiosalicylate for absolute peptide and protein quantification.

The methylmercury ion (CH(3)Hg(+)) demonstrated a high efficiency for directly labeling peptide/protein based on its specific and strong interaction with the sulfhydryl(s) in the peptide/protein and because of its smallest size among monofunctional organic mercurials studied, including methylmercury, ethylmercury, 4-(hydroxymercuric)benzoic acid, and 2,7-dibromo-4-hydroxymercurifluoresceine disodium. A simple 1:1 stoichiometry between CH(3)Hg(+) and sulfhydryl, confirmed with electrospray ionization-mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) studies, made it easy to calibrate the stoichiometry of Hg in the peptide/protein. In order to avoid the direct use of the harmful CH(3)Hg(+), in this study a CH(3)Hg(+)-equivalent tag, methylmercurithiosalicylate (CH(3)Hg-THI), and its (204)Hg-enriched homologue (CH(3)(204)Hg-THI) were synthesized, and then CH(3)Hg(+) and/or CH(3)(204)Hg(+) released from CH(3)Hg-THI and/or CH(3)(204)Hg-THI in solution were utilized to demonstrate the dynamic labeling of glutathione (GSH) and two model proteins, beta-lactoglobulin (BLG) and ovalbumin (OVA), for the first time. Furthermore, the CH(3)(204)Hg-THI isotopical labeled GSH, BLG, and OVA standards (CH(3)(204)Hg-GSH, CH(3)(204)Hg-BLG, and CH(3)(204)Hg-OVA) were used to demonstrate the feasibility of absolute peptide/protein quantification using label-specific isotope dilution inductively coupled plasma mass spectrometry (ICPMS). On the basis of the accurate and sensitive determination of Hg using ICPMS, the detection limits of GSH, BLG, and OVA could reach 45.4, 45.4, and 15.1 pmol L(-1), respectively, suggesting the possibility for low-abundance peptide/protein quantification alongside the surefire quantification of moderate and highly abundant peptide/protein.

Organometallic activation of a fluorogen for templated nucleic acid detection.

A nucleic acid detection scheme that employs DNA-mediated delivery of an organomercury activator to unmask a fluorophore is described. The approach relies on adjacent hybridization of two oligonucleotide conjugates containing organomercury and caged rhodamine functionalities. Postsynthetic conjugation of amino-modified DNAs enabled efficient preparation of these probes. Complementary DNA templates yielded fluorescence signals arising from metal-assisted rhodamine uncaging.

Enrichment by organomercurial agarose and identification of cys-containing peptides from yeast cell lysates.

Dynamic range and the presence of highly abundant proteins limit the number of proteins that may be identified within a complex mixture. Cysteine (Cys) has unique chemical reactivity that may be exploited for chemical tagging/capture with biotin/avidin reagents or affinity chromatography allowing specific isolation and subsequent identification of peptide sequences by mass spectrometry. Organomercurial agarose (Hg-beads) specifically captures Cys-containing peptides and proteins from cell lysates. Tryptic peptides from yeast lysates containing Cys were captured and eluted from Hg-beads after incubation with TCEP and trypsin. From two 1 h nano 1-D LC DDA/MS of the eluate >700 proteins were identified with an estimated false positive rate of approximately 1%. Few peptides were identified with high confidence without Cys within their sequence after capture, and extensive washing, indicating little nonspecific binding. The number of fragmentation spectra was increased using automated 2-D nano-LC/MS and allowed identification of 1496 proteins with an estimated false positive rate of 1.1%. Approximately 4% of the proteins identified were from peptides that did not contain Cys, and these were biased toward higher abundance proteins. Comparison of the 1496 proteins to those reported previously showed that >25% were from yeast proteins not previously observed. Most proteins were identified from a single peptide, and sequence coverage was sacrificed by focusing only on identifying Cys-containing peptides, but large numbers of proteins were rapidly identified by eliminating many of the peptides from the higher abundance proteins.

Cleaving mercury-alkyl bonds: a functional model for mercury detoxification by MerB.

The extreme toxicity of organomercury compounds that are found in the environment has focused attention on the mechanisms of action of bacterial remediating enzymes. We describe facile room-temperature protolytic cleavage by a thiol of the Hg-C bond in mercury-alkyl compounds that emulate the structure and function of the organomercurial lyase MerB. Specifically, the tris(2-mercapto-1-t-butylimidazolyl)hydroborato ligand [Tm(Bu(t))], which features three sulfur donors, has been used to synthesize [Tm(Bu(t))]HgR alkyl compounds (R = methyl or ethyl) that react with phenylthiol (PhSH) to yield [Tm(Bu(t))]HgSPh and RH. Although [Tm(Bu(t))]HgR compounds exist as linear two-coordinate complexes in the solid state, 1H nuclear magnetic resonance spectroscopy indicates that the complexes exist in rapid equilibrium with their higher-coordinate [kappa2-Tm(Bu(t))]HgR and [kappa3-Tm(Bu(t))]HgR isomers in solution. Facile access to a higher-coordinate species is proposed to account for the exceptional reactivity of [Tm(Bu(t))]HgR relative to that of other two-coordinate mercury-alkyl compounds.

Mercury(II) 2-aminoethanethiolate clusters: intramolecular transformations and mechanisms.

The combination of HgF2 and 2-aminoethanethiol (AET, with some AET.HCl present) yielded a cyclic tetranuclear thiolate, [Hg4Cl4(SCH2CH2NH2)4] (1), with alternating Hg and S atoms. The Cl from the reaction mixture led to the formation of Hg-Cl bonds with no Hg-F in the final product. In contrast, a similar reaction with HgBr2 yielded a nonanuclear cluster, [Hg9Br15(SCH2CH2NH3)15]3+ (2), and the disulfide salt {[HgBr4][(NH3CH2CH2S-)2]} (3). Despite similar reactions, the AET groups in 2 are protonated compared to the nonprotonated amine groups in 1, which allows the ligand to chelate the Hg atom in the latter compound. The reaction with HgI2 yielded a cyclic tetranuclear compound, [Hg4I6(SCH2CH2NH2)2(SCH2CH2NH3)2](H2O/EtOH) (4), containing protonated and nonprotonated AET groups. Compound 4 at room temperature irreversibly rearranges to [Hg4I4(SCH2CH2NH2)4] (5), which is isostructural to 1. A systematic pathway for the formation of 1 along with the intramolecular conversion of 4 to 5 is proposed. These compounds demonstrate that very diverse Hg-S compounds form under similar reaction conditions.

[Assay of three kinds of aluminum fractions (Al(a), Al(b) and Al(c)) in polynuclear aluminum solutions by Al-Ferron timed spectrophotometry and demarcation of their time limits].

Al-Ferron timed spectrophotometry assay is a basic method in the study on the formation of polynuclear hydroxyl aluminum species and their transformation laws in aqueous systems. In actual working process, this methodology has some dogmatism and arbitrariness in the time limits demarcation of the three kinds of aluminum fractions (Al(a), Al(b) and Al(c)) in polynuclear aluminum solutions, which makes this kind of classification rougher, and the experimental results non-reproducible. The reason for this difference is that the specific species within Al(a), Al(b) and Al(c) have different reaction mechanism and dynamics, and that specific species of Al(b) having different OH/Al ratios have different reaction rates with ferron. In this paper, the ExpAssoc distribution was applied to quantitatively fit the Al-Ferron reaction dynamics curve, and the extrapolation method was used to survey the 1 min measured value [Al(a)] of monomeric Al, which is hard to obtain in manual manipulation. The time demarcation between Al(b) and Al(c) should reach the point of the experimental data curve up to horizontal platform. The microwave-radiated technology was used to fast assay the total aluminum concentration [Al(T)]. With these methods, the contents of monomeric Al(a), polynuclear Al(b) and gel Al(c) can be conveniently and quantitatively measured. It offers a novel method for surmounting the arbitrariness in the measurement of the three kinds of aluminum fractions and the repetitive calculation of Al(a) and Al(b).

Inside or outside a ligand cleft? Synthetic, structural, and kinetic inertness studies of zinc, cadmium, and mercury complexes of cross-bridged cyclam and cyclen.

Ethylene cross-bridging of the popular tetraazamacrocyclic ligand cyclam has led to metal complexes with enhanced kinetic inertness. The synthesis and spectral characterization of zinc(II), cadmium(II), and mercury(II) complexes of cross-bridged cyclam (L1) as well as cross-bridged cyclen (L2) are reported along with the details of our synthetic route to L2. X-ray structural studies revealed that all Zn(II) and Cd(II) cations are fully kappa(4)N-coordinated inside the respective ligand's molecular cleft with L1 providing the better fit for Zn(II). While Hg(II) is similarly coordinated to L2, it has been found to complex L1 outside the ligand cleft in a novel exo-kappa(2)N-mode. Solution NMR data of the kappa(4)N complexes are consistent with the presence of only a single cis-folded isomer in each case. Ligand (1)H and (13)C coupling to both (111,113)Cd and (199)Hg in their complexes can be clearly discerned. The relative kinetic inertness of representative cross-bridged complexes in acidic aqueous solution has been assessed and found to be in the following order: Zn(II) > Cd(II)[dbl greater-than] Hg(II). The data also reaffirm that cross-bridged cyclam ligand L1 forms a substantially more inert complex with zinc(II) than either the smaller cyclen analogue L2 or the unbridged 1,4,8,11-tetramethyl-cyclam L3.

NMR structural studies reveal a novel protein fold for MerB, the organomercurial lyase involved in the bacterial mercury resistance system.

Mercury resistant bacteria have developed a system of two enzymes (MerA and MerB), which allows them to efficiently detoxify both ionic and organomercurial compounds. The organomercurial lyase (MerB) catalyzes the protonolysis of the carbon-mercury bond resulting in the formation of ionic mercury and a reduced hydrocarbon. The ionic mercury [Hg(II)] is subsequently reduced to the less reactive elemental mercury [Hg(0)] by a specific mercuric reductase (MerA). To better understand MerB's unique enzymatic activity, we used nuclear magnetic resonance (NMR) spectroscopy to determine the structure of the free enzyme. MerB is characterized by a novel protein fold consisting of three noninteracting antiparallel beta-sheets surrounded by six alpha-helices. By comparing the NMR data of free MerB and the MerB/Hg/DTT complex, we identified a set of residues that likely define a Hg/DTT binding site. These residues cluster around two cysteines (C(96) and C(159)) that are crucial to MerB's catalytic activity. A detailed analysis of the structure revealed the presence of an extensive hydrophobic groove adjacent to this Hg/DTT binding site. This extensive hydrophobic groove has the potential to interact with the hydrocarbon moiety of a wide variety of substrates and may explain the broad substrate specificity of MerB.

Organomercury bioconjugate synthesis and characterization by matrix-assisted laser desorption ionization mass spectrometry.

Synthesis of an organomercury hapten and conjugation of the hapten to proteins and peptides is described. Starting with allylamine, synthesis of the organomercury hapten was completed in five steps using readily available and inexpensive reagents. The key transformation in the synthesis, intramolecular oxymercuration, was achieved in good yield and under mild conditions. Hapten conjugation was afforded via disuccinimide active ester coupling chemistry, and the resulting conjugates were analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). To exploit the accurate mass measuring capabilities of MALDI-MS, the conjugates were digested with trypsin prior to analysis. The masses of the peptides resulting from tryptic digestion of the organomercury conjugates were accurately measured, and five hapten attachments were identified in the mass range of 1000-2200 m/z. The organomercury bioconjugate synthesized in this study was designed to contain a stable carbon-metal bond, constituting an underutilized approach for preparing protein-metal complexes and may result in mAbs consisting of unique recognition capabilities.

Phytoremediation of organomercurial compounds via chloroplast genetic engineering.

Mercury (Hg), especially in organic form, is a highly toxic pollutant affecting plants, animals, and man. In plants, the primary target of Hg damage is the chloroplast; Hg inhibits electron transport and photosynthesis. In the present study, chloroplast genetic engineering is used for the first time to our knowledge to enhance the capacity of plants for phytoremediation. This was achieved by integrating a native operon containing the merA and merB genes (without any codon modification), which code for mercuric ion reductase (merA) and organomercurial lyase (merB), respectively, into the chloroplast genome in a single transformation event. Stable integration of the merAB operon into the chloroplast genome resulted in high levels of tolerance to the organomercurial compound, phenylmercuric acetate (PMA) when grown in soil containing up to 400 micro M PMA; plant dry weights of the chloroplast transformed lines were significantly higher than those of wild type at 100, 200, and 400 micro M PMA. That the merAB operon was stably integrated into the chloroplast genome was confirmed by polymerase chain reaction and Southern-blot analyses. Northern-blot analyses revealed stable transcripts that were independent of the presence or absence of a 3'-untranslated region downstream of the coding sequence. The merAB dicistron was the more abundant transcript, but less abundant monocistrons were also observed, showing that specific processing occurs between transgenes. The use of chloroplast transformation to enhance Hg phytoremediation is particularly beneficial because it prevents the escape of transgenes via pollen to related weeds or crops and there is no need for codon optimization to improve transgene expression. Chloroplast transformation may also have application to other metals that affect chloroplast function.

The roles of thiols in the bacterial organomercurial lyase (MerB).

The bacterial plasmid-encoded organomercurial lyase, MerB (EC 4.99.1.2), catalyzes the protonolysis of organomercury compounds yielding Hg(II) and the corresponding protonated hydrocarbon. A small, soluble protein with no known homologues, MerB is widely distributed among eubacteria in three phylogenetically distinct subfamilies whose most prominent motif includes three conserved cysteine residues. We found that the 212-residue MerB encoded by plasmid R831b is a cytosolic enzyme, consistent with its high thiol requirement in vitro. MerB is inhibited by the nonphysiological dithiol DTT but uses the physiological thiols, glutathione and cysteine, equally well. Highly conserved Cys96 and Cys159 are essential for activity, whereas weakly conserved Cys160 is not. Proteins mutant in highly conserved Cys117 are insoluble. All MerB cysteines are DTNB-reactive in native and denatured states except Cys117, which fails to react with DTNB in the native form, suggesting it is buried. Mass spectrometric analysis of trypsin fragments of reduced proteins treated with N-ethylmaleimide or iodoacetamide revealed that all cysteines form covalent adducts and remain covalently modifiable even when exposed to 1:1 PHMB prior to treatment with NEM or IAM. Stable PHMB adducts were also observed on all cysteines in mutant proteins, suggesting rapid exchange of PHMB among the remaining protein thiols. However, PHMB exposure of reduced wild-type MerB yielded only Hg adducts on the Cys159/Cys160 peptide, suggesting a trapped reaction intermediate. Using HPLC to follow release of benzoic acid from PHMB, we confirmed that fully reduced wild-type MerB and mutant C160S can carry out a single protonolysis without exogenous thiols. On the basis of the foregoing we refine the previously proposed S(E)2 mechanism for protonolysis by MerB.