Functional evaluation of Asp76, 84, 102 and 150 in human arsenic(III) methyltransferase (hAS3MT) interacting with S-adenosylmethionine

AbstractWe prepared eight mutants (D76P, D76N, D84P, D84N, D102P, D102N, D150P and D150N) to investigate the functions of residues Asp76, 84, 102 and 150 in human arsenic(III) methyltransferase (hAS3MT) interacting with the S-adenosylmethionine (SAM)-binding. The affinity of all the mutants for SAM were weakened. All the mutants except for D150N completely lost their methylation activities. Residues Asp76, 84, 102 and 150 greatly influenced hAS3MT catalytic activity via affecting SAM-binding or methyl transfer. Asp76 and 84 were located in the SAM-binding pocket, and Asp102 significantly affected SAM-binding via forming hydrogen bonds with SAM

Soil N-oxide emissions decrease from intensive greenhouse vegetable fields by substituting synthetic N fertilizer with organic and bio-organic fertilizers

In order to reduce soil and environmental quality degradation associated with the use of synthetic nitrogen (N), substituting chemical fertilizer with organic or bio-organic fertilizer has become an increasingly popular option. However, components of this fertilizer strategy related to mitigation of soil N-oxide emissions and maintenance of crop yield remain uncertain. Here, we evaluated the effects of three different fertilizer strategies, with equal amounts of N, on nitrous oxide (N2O) and nitric oxide (NO) emissions, vegetable yield, and yield-scaled N2O and NO emissions under three consecutive cucumber growing seasons. The three treatments were chemical fertilizer (NPK, urea), organic fertilizer (O, composted cattle manure), and bio-organic fertilizer (O + T, O combined with Trichoderma.spp). Results showed that the NPK plot had the highest area-scaled emissions of N2O (13.1 ± 0.48 kg N ha−1 yr−1) and NO (5.01 ± 0.34 kg N ha−1 yr−1), which were 1.3–1.4 and 3.1–3.7 times greater than the O and O + T plots, respectively. The annual direct emission factors for N2O and NO were 2.08% and 0.92% for the NPK plot, which declined to 1.34% and 0.09% in the O plot, and 1.12% and 0.03% in the O + T plot, respectively. The annual vegetable yield was 117 ± 2.9 t ha−1 for NPK plot and 122 ± 2.0 t ha−1 for O + T plot, which was higher than 111 ± 1.7 t ha−1 for O plot. The yield-scaled N2O + NO emissions differed significantly with fertilization treatment, with the lowest value observed in the O + T plot. We attributed the lower soil N-oxide emissions following organic fertilizer application to the slow release of available N and enhanced denitrification caused by the increase of soil dissolved organic carbon and pH. Compared with the use of organic fertilizer alone, the addition of Trichoderma.spp significantly increased the potential denitrification rate but decreased N2O emissions, which may have promoted the reduction of N2O to N2. Therefore, our results suggest that adopting composted organic fertilizer mixtures with microbial inoculants could be a win-win practice to mitigate gaseous N losses and simultaneously improve crop yield in intensively managed vegetable cropping systems

The functions of crucial cysteine residues in the arsenite methylation catalyzed by recombinant human arsenic (III) methyltransferase.

Arsenic (III) methyltransferase (AS3MT) is a cysteine (Cys)-rich enzyme that catalyzes the biomethylation of arsenic. To investigate how these crucial Cys residues promote catalysis, we used matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to analyze Cys residues in recombinant human arsenic (III) methyltransferase (hAS3MT). We detected two disulfide bonds, Cys250-Cys32 and Cys368-Cys369, in hAS3MT. The Cys250-Cys32 disulfide bond was reduced by glutathione (GSH) or other disulfide bond reductants before the enzymatic methylation of arsenite (iAs3+). In addition to exposing residues around the active sites, cleavage of the Cys250-Cys32 pair modulated the conformation of hAS3MT. This adjustment may stabilize the binding of S-Adenosyl-L-methionine (AdoMet) and favor iAs3+ binding to hAS3MT. Additionally, we observed the intermediate of Cys250-S-adenosylhomocysteine (AdoHcy), suggesting that Cys250 is involved in the transmethylation. In recovery experiments, we confirmed that trivalent arsenicals were substrates for hAS3MT, methylation of arsenic occurred on the enzyme, and an intramolecular disulfide bond might be formed after iAs3+ was methylated to dimethylarsinous acid (DMA3+). In this work, we clarified both the functional roles of GSH and the crucial Cys residues in iAs3+ methylation catalyzed by hAS3MT

Tetraarsenictetrasulfide and Arsenic Trioxide Exert Synergistic Effects on Induction of Apoptosis and Differentiation in Acute Promyelocytic Leukemia Cells.

Since arsenic trioxide (As3+) has been successfully used in the treatment of acute promyelocytic leukemia (APL), its adverse effects on patients have been problematic and required a solution. Considering the good therapeutic potency and low toxicity of tetraarsenictetrasulfide (As4S4) in the treatment of APL, we investigated the effects of combining As4S4 and As3+ on the apoptosis and differentiation of NB4 and primary APL cells. As4S4, acting similarly to As3+, arrested the G1/S transition, induced the accumulation of cellular reactive oxygen species, and promoted apoptosis. Additionally, low concentrations of As4S4 (0.1-0.4 μM) induced differentiation of NB4 and primary APL cells. Compared with the As4S4- or As3+-treated groups, the combination of As4S4 and As3+ obviously promoted apoptosis and differentiation of NB4 and primary APL cells. Mechanistic studies suggested that As4S4 acted synergistically with As3+ to down-regulate Bcl-2 and nuclear factor-κB expression, up-regulate Bax and p53 expression, and induce activation of caspase-12 and caspase-3. Moreover, the combination of low concentrations of As4S4 and As3+ enhanced degradation of the promyelocytic leukemia-retinoic acid receptor α oncoprotein. In summary, As4S4 and As3+ synergistically induce the apoptosis and differentiation of NB4 and primary APL cells

Optimisation of An HHG-Seeded Harmonic Cascade FEL Design for the NLS Project

Optimization studies of an HHG-seeded harmonic cascade FEL design for the UK's proposed New Light Source (NLS) facility are presented. Three separate FELs are planned to meet the requirements for continuous coverage of the photon energy range 50-1000 eV with variable polarization, 20 fs pulse widths and good temporal coherence. The design uses an HHG seed source tuneable from 50-100 eV to provide direct FEL seeding in this range, and one or two stage harmonic cascades to reach the higher photon energies. Studies have been carried out to optimize a harmonic cascade FEL operating at 1 keV; topics investigated include modulator configuration, seed power level and ef- fects of the HHG seed structure. FEL simulations using realistic electron beam distributions are presented and tolerance to increased emittance has been considered

Identification of the third binding site of arsenic in human arsenic (III) methyltransferase.

Arsenic (III) methyltransferase (AS3MT) catalyzes the process of arsenic methylation. Each arsenite (iAs(3+)) binds to three cysteine residues, methylarsenite (MMA(3+)) binds to two, and dimethylarsenite (DMA(3+)) binds to one. However, only two As-binding sites (Cys156 and Cys206) have been confirmed on human AS3MT (hAS3MT). The third As-binding site is still undefined. Residue Cys72 in Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) may be the third As-binding site. The corresponding residue in hAS3MT is Cys61. Functions of Cys32, Cys61, and Cys85 in hAS3MT are unclear though Cys32, Cys61, and Cys85 in rat AS3MT have no effect on the enzyme activity. This is why the functions of Cys32, Cys61, and Cys85 in hAS3MT merit investigation. Here, three mutants were designed, C32S, C61S, and C85S. Their catalytic activities and conformations were determined, and the catalytic capacities of C156S and C206S were studied. Unlike C85S, mutants C32S and C61S were completely inactive in the methylation of iAs(3+) and active in the methylation of MMA(3+). The catalytic activity of C85S was also less pronounced than that of WT-hAS3MT. All these findings suggest that Cys32 and Cys61 markedly influence the catalytic activity of hAS3MT. Cys32 and Cys61 are necessary to the first step of methylation but not to the second. Cys156 and Cys206 are required for both the first and second steps of methylation. The S(C32) is located far from arsenic in the WT-hAS3MT-SAM-As model. The distances between S(C61) and arsenic in WT-hAS3MT-As and WT-hAS3MT-SAM-As models are 7.5 Å and 4.1 Å, respectively. This indicates that SAM-binding to hAS3MT shortens the distance between S(C61) and arsenic and promotes As-binding to hAS3MT. This is consistent with the fact that SAM is the first substrate to bind to hAS3MT and iAs is the second. Model of WT-hAS3MT-SAM-As and the experimental results indicate that Cys61 is the third As-binding site

Residues in human arsenic (+3 oxidation state) methyltransferase forming potential hydrogen bond network around S-adenosylmethionine.

Residues Tyr59, Gly78, Ser79, Met103, Gln107, Ile136 and Glu137 in human arsenic (+3 oxidation state) methyltransferase (hAS3MT) were deduced to form a potential hydrogen bond network around S-adenosylmethionine (SAM) from the sequence alignment between Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) and hAS3MT. Herein, seven mutants Y59A, G78A, S79A, M103A, Q107A, I136A and E137A were obtained. Their catalytic activities and conformations were characterized and models were built. Y59A and G78A were completely inactive. Only 7.0%, 10.6% and 13.8% inorganic arsenic (iAs) was transformed to monomethylated arsenicals (MMA) when M103A, Q107A and I136A were used as the enzyme. The Vmax (the maximal velocity of the reaction) values of M103A, Q107A, I136A and E137A were decreased to 8%, 22%, 15% and 50% of that of WT-hAS3MT, respectively. The KM(SAM) (the Michaelis constant for SAM) values of mutants M103A, I136A and E137A were 15.7, 8.9 and 5.1 fold higher than that of WT-hAS3MT, respectively, indicating that their affinities for SAM were weakened. The altered microenvironment of SAM and the reduced capacity of binding arsenic deduced from KM(As) (the Michaelis constant for iAs) value probably synergetically reduced the catalytic activity of Q107A. The catalytic activity of S79A was higher than that of WT despite of the higher KM(SAM) , suggesting that Ser79 did not impact the catalytic activity of hAS3MT. In short, residues Tyr59 and Gly78 significantly influenced the catalytic activity of hAS3MT as well as Met103, Ile136 and Glu137 because they were closely associated with SAM-binding, while residue Gln107 did not affect SAM-binding regardless of affecting the catalytic activity of hAS3MT. Modeling and our experimental results suggest that the adenine ring of SAM is sandwiched between Ile136 and Met103, the amide group of SAM is hydrogen bonded to Gly78 in hAS3MT and SAM is bonded to Tyr59 with van der Waals, cation-π and hydrogen bonding contacts