MAT Activity Research Articles

Preservational modes of some ichthyosaur soft tissues (Reptilia, Ichthyopterygia) from the Jurassic Posidonia Shale of Germany

AbstractKonservat‐Lagerstätten, such as the Toarcian (Early Jurassic) Posidonia Shale of southwestern Germany, are renowned for their spectacular fossils. Ichthyosaur skeletons recovered from this formation are frequently associated with soft tissues; however, the preserved material ranges from three‐dimensional, predominantly phosphatized structures to dark films of mainly organic matter. We examined soft‐tissue residues obtained from two ichthyosaur specimens using an integrated ultrastructural and geochemical approach. Our analyses revealed that the superficially‐looking ‘films’ in fact comprise sections of densely aggregated melanosome (pigment) organelles sandwiched between phosphatized layers containing fibrous microstructures. We interpret this distinct layering as representing condensed and incompletely degraded integument from both sides of the animal. When compared against previously documented ichthyosaur fossils, it becomes readily apparent that a range of preservational modes exists between presumed ‘phosphatic’ and ‘carbonized’ soft‐tissue remains. Some specimens show high structural fidelity (e.g. distinct integumentary layering), while others, including the fossils examined in this study, retain few original anatomical details. This diversity of soft‐tissue preservational modes among Posidonia Shale ichthyosaurs offers a unique opportunity to examine different biostratinomic, taphonomic and diagenetic variables that potentially could affect the process of fossilization. It is likely that soft‐tissue preservation in the Posidonia Shale was regulated by a multitude of factors, including decay efficiency and speed of phosphatic mineral nucleation; these in turn were governed by a seafloor with sustained microbial mat activity fuelled by high organic matter input and seasonally fluctuating oxygen levels.

Detectable reentrant circuits in localized microreentrant tachycardias in the era of ultra high-density mapping

Abstract Funding Acknowledgements Type of funding sources: None. Background Optimal criteria in clinical practice differentiating microreentrant atrial tachycardias (mAT) from macroreentrant atrial arrhythmias (MAT) have not yet been clarified. The use of multielectrode catheters and the recent development of ultra high-density mapping (UHDM) with automated features has improved our knowledge of mAT circuits and foci location. Purpose In the present study, we systematically evaluated AT mechanisms in a large consecutive cohort of patients with MAT identifying the precise mAT circuits using the Rhythmia mapping system. Methods Consecutive patients indicated for MAT ablation from January 2021 to November 2021 at 33 centers were prospectively included. All MATs were completely mapped in the left or right atrium by means of the Rhythmia mapping system and the 64-poles Orion basket catheter. For study purpose, a mAT was defined as an AT with slow continuous low fragmented potentials covering at least 50% of tachycardia cycle length (CL) in a small area (set as a circuit within < 1 cm2) and in a couple of closed splines of the Orion catheter and a centrifugal activation pattern to the remainder of the atria. The Lumipoint tool was systematically used to confirm EGM fragmentation inside this area. Data are reported as mean±SD. Results One-hundred eighty-seven MATs were analyzed: 100 (53.7%) atypical left atrial flutter, 27 (14.3%) left AT and 60 (32%) right AT. A total of 7 MAT (prevalence of 3.7%) was identified as mATs (6 atypical left atrial flutter and 1 AT), with 5 out 7 with a previous history of AF ablation procedure. The percentage of atrial surface with a voltage level below 0.1 mV was 19±17%. The CL was 329±78ms. The electrical activity spanning the whole CL was detected by 2±0.6 pairs of close bipoles of the Orion catheter, and was actually confined to a region of 0.4±0.2cm2 with continuous highly fractionated potential covering 68±10% of the CL (longest component of fractionated EGM per spline=74±18% of the CL). Voltage level was 0.3±0.1mV at RF delivery site and 0.2±0.1mV at the site of longest duration of the fragmented potential, respectively. Targeted mAT activity was identified closer to PVs in three cases and at the mid portion of the anterior wall and at the roof in two cases each, respectively. In all cases a single shot RF delivery terminated each arrhythmia at targeted location. Consolidative RF ablations were then delivered in the adjacent area. No complication occurred. At three months follow-up all patients remained free from any AT recurrence. Conclusions In this standard of care clinical experience with UHDM system, the prevalence of mAT seems to be higher than previously reported in literature. A technique based on mAT identification through a novel automated algorithm and matched area of electrogram fractionation captured by the Orion catheter may limit the extent of ablation needed.

Ontogeny of hepatic methionine catabolic enzyme activities (Transmethylation and Transsulphuration) and associated physiological amino acids in E10-21 chick embryos and D1-49 broilers.

Developmental changes in hepatic methionine adenosyltransferase, cystathionine β-synthase, cystathionase, and glycine N-methyltransferase were determined in broiler chick embryos and hatched chicks by using radiometric and spectrometric methods. Hepatic free methionine, S-adenosylmethionine, S-adenosylhomocysteine, homocysteine, cystathionine, and cysteine levels were also investigated. Results showed an increase in hepatic MAT activity from E10 to E21 during embryogenesis, suggesting greater transmethylation rates throughout the rapid embryonic growth and development period. A strong positive correlation between embryo BW and MAT activity also supports this idea. The MAT specific activity continued to increase after hatching, but there was a negative correlation between chick BW and MAT activities from D1 to D49. This may indicate different MAT isozymes exist for chick embryo hepatic tissue compared to hepatic tissue of hatched chick and growing broilers. The developmental pattern of MAT isozymes could be critical for methionine metabolism to cope with the demand imposed on the embryo, chicks, and growing broilers. Additionally, the specific activity of hepatic CBS in chick embryos was determined to be lower compared to that observed in older broilers (35 and 49days). Since liver CBS specific activity is at the lowest point from D1-7 in young chicks, the ability to convert adequate homocysteine to cysteine through transsulphuration may be limiting for cysteine synthesis at this time. Steady-state hepatic homocysteine levels in chick embryos and chicks may be a function of the rates of homocysteine formation, remethylation, and catabolism via the transsulphuration pathway. The present study indicates young chicks from D1 to D7 may have a limited ability for adequate transsulphuration; therefore, dietary cystine may be needed for optimum performance.

Detection and community-level identification of microbial mats in the McMurdo Dry Valleys using drone-based hyperspectral reflectance imaging

AbstractThe reflectance spectroscopic characteristics of cyanobacteria-dominated microbial mats in the McMurdo Dry Valleys (MDVs) were measured using a hyperspectral point spectrometer aboard an unmanned aerial system (remotely piloted aircraft system, unmanned aerial vehicle or drone) to determine whether mat presence, type and activity could be mapped at a spatial scale sufficient to characterize inter-annual change. Mats near Howard Glacier and Canada Glacier (ASPA 131) were mapped and mat samples were collected for DNA-based microbiome analysis. Although a broadband spectral parameter (a partial normalized difference vegetation index) identified mats, it missed mats in comparatively deep (> 10 cm) water or on bouldery surfaces where mats occupied fringing moats. A hyperspectral parameter (B6) did not have these shortcomings and recorded a larger dynamic range at both sites. When linked with colour orthomosaic data, B6 band strength is shown to be capable of characterizing the presence, type and activity of cyanobacteria-dominated mats in and around MDV streams. 16S rRNA gene polymerase chain reaction amplicon sequencing analysis of the mat samples revealed that dominant cyanobacterial taxa differed between spectrally distinguishable mats, indicating that spectral differences reflect underlying biological distinctiveness. Combined rapid-repeat hyperspectral measurements can be applied in order to monitor the distribution and activity of sentinel microbial ecosystems across the terrestrial Antarctic.

Sobre el vínculo afectivo y los procesos de aprendizaje en niños de 7 a 9 años de edad

Objetivo: comprender la incidencia de la ruptura del vínculo afectivo en el proceso de aprendizaje del niño, suceso que lleva a la separación de dos personas entre las que se encuentra establecida una relación afectiva. Método: este estudio se basó en un paradigma cualitativo, con un enfoque histórico hermenéutico; y de la misma forma, contó con resultados obtenidos de las técnicas de recolección de información tales como: entrevistas, observaciones participantes a través de una actividad de colcha de retazos, talleres y análisis documental. Resultados: a través de esta investigación se determinó la importancia del vínculo afectivo durante el desarrollo de los niños, hecho percibido en el poco tiempo de calidad brindando por los padres a sus hijos. Además, esto llevó a generar dificultades en el aprendizaje de los escolares, conllevando a un proceso académico regular. Discusión y Conclusiones: Se discurre que el rompimiento del vínculo afectivo afecta de manera diferente el desarrollo de los niños y niñas, ya que a pesar de que suelen superar el rompimiento o parecen hacerlo, en el taller realizado se evidenció que hay ciertos estudiantes que podrían tener una mayor afectación en su proceso de aprendizaje, esto debido a los diferentes factores que han tenido que sobrellevar, por lo que el rompimiento de su vínculo ha sido más traumático. Por consiguiente, se concluyó a partir del proceso sistemático planteado que, el vínculo afectivo se da a través del compartir mutuo y la relaciones calidad entre dos personas

S-adenosylmethionine synthases in plants: Structural characterization of type I and II isoenzymes from Arabidopsis thaliana and Medicago truncatula

S-adenosylmethionine synthases (MATs) are responsible for production of S-adenosylmethionine, the cofactor essential for various methylation reactions, production of polyamines and phytohormone ethylene, etc. Plants have two distinct MAT types (I and II). This work presents the structural analysis of MATs from Arabidopsis thaliana (AtMAT1 and AtMAT2, both type I) and Medicago truncatula (MtMAT3a, type II), which, unlike most MATs from other domains of life, are dimers where three-domain subunits are sandwiched flat with one another. Although MAT types are very similar, their subunits are differently oriented within the dimer. Structural snapshots along the enzymatic reaction reveal the exact conformation of precatalytic methionine in the active site and show a binding niche, characteristic only for plant MATs, that may serve as a lock of the gate loop. Nevertheless, plants, in contrary to mammals, lack the MAT regulatory subunit, and the regulation of plant MAT activity is still puzzling. Our structures open a possibility of an allosteric activity regulation of type I plant MATs by linear compounds, like polyamines, which would tighten the relationship between S-adenosylmethionine and polyamine biosynthesis.

Distribution, redox state and (bio)geochemical implications of arsenic in present day microbialites of Laguna Brava, Salar de Atacama

Understanding how microorganisms adapted to the high arsenic concentration present on early Earth requires understanding of the processes involved in the arsenic biogeochemical cycle operating in living microbial mats. To this end, we investigated a living microbial mat from Laguna Brava (Salar de Atacama, Chile), a hypersaline lake with high arsenic concentration, using an array of conventional geochemical techniques, such as X-ray diffraction, SEM-EDX and Confocal Laser Scanning Microscopy (CLSM), combined with state-of-the-art high resolution scanning imaging techniques, including X-ray micro-fluorescence (μXRF) and X-ray Absorption Near Edge Structure (XANES) mapping. This experimental approach allowed us to unravel the relationship between the microbial mat activity, mineral occurrence, arsenic speciation, distribution of major and trace elements and their relationship with the mineralogy and the exopolymeric substances (EPS). We show that As was not linked to Ca or Si, and only moderately related to Fe, resulting from sorption onto an iron (oxy)hydroxide mineral. In addition, we were able to identify organic-rich globules containing significant As but no other trace metal(loid)s, and determine the co-existence of As(III) and As(V). These observations strongly support the occurrence of microbially-mediated arsenic cycling in these microbial mats.

Electrons from Solid Electrode Alter Phototrophic Mat Morphology and Metabolic Activity

Phototrophic microbial mats are structured microbial communities composed of highly interactive species that have complete energy and elements within them. Aside from being an excellent ecological model that can be used to investigate how microbial populations associate and interact, microbial mats have the potential for energy capture and conversion applications. To use mats for such purpose, it is critical to understand the energy transfer and activity of microbial populations under the existence of external electron donor or acceptor sources. One method to accomplish this is by introducing a solid electrode with a controlled electrochemical potential to act as an electron donor or acceptor to the mat systems. We hypothesized that varying the electrode potential would, in turn, change the structure, activity, and community composition of the mat via regulation of electron transfer processes. In this work, the electrodes were placed under mats to establish a mat electrochemical system for testing our hypothesis. The phototrophic microbial mat was harvested from Hot Lake, a hypersaline, epsomitic lake located near Oroville (Washington, USA). The electrodes in contact with the base of the mat were polarized at -0.7 VAg/AgCl (hereafter referred to as the “cathodic mat)”. Electrodes that served as an electron sink were polarized at +0.3 VAg/AgCl (hereafter referred as the “anodic mat”). We quantified the diel variations of electron transfer rates between the electrode and the cathodic or anodic mats. We observed variations in current over a diel light cycle in which the lowest and highest magnitudes of anodic current occurred simultaneously with those of cathodic current. To understand the factors affecting these variations we examined the influences of temperature, light intensity, and substrate additions, separately, to electron transfer rate. We also used microelectrodes to measure the depth profiles of sulfide and oxygen concentrations and compared them with the variations of electron transfer rates over a diel cycle. To understand how anodic or cathodic electron transfer processes impacted mat structure and activity, we used nuclear magnetic resonance (NMR) imaging for morphology and NMR metabolite analysis to quantify the activity. The results showed that the quantitative porosity and diffusion coefficients were higher in the cathodic mat than in the anodic mat. In addition, cathodic mats produced significantly higher concentrations of osmo-protectants such as betaine and trehalose. The anodic mat had metabolic profiles similar to the control (non-polarized) mat. Finally, we employed amplicon sequencing across the V4 region of the 16S gene to determine the effect of the electrochemical regime upon mat community structure. The community structure data were consistent with variations in metabolite profiles and suggested greater changes in microbial community structure and function in the cathodic vs. the anodic and control mats. Although further work is needed to determine whether the electron transfer to and from the electrode to the mat was mediated by microbial metabolism of selectively-enriched community on the electrode or was caused by abiotic reactions on electrode, our data strongly suggest that donation of electrons via a solid electrode is capable of altering phototrophic mat morphology, community composition, and metabolic activity. It is possible that the addition of electrons from electrode to the mat alleviated the energy limitations allowing the mat to maintain large carbon pools of osmolytes. In addition, some microorganisms in the mat might use electrons from the electrode to drive carbon fixations which resulted in selectively-enriched community on the electrode and the increase of the total carbon available to the mat.

Regulation of electron transfer processes affects phototrophic mat structure and activity.

Phototrophic microbial mats are among the most diverse ecosystems in nature. These systems undergo daily cycles in redox potential caused by variations in light energy input and metabolic interactions among the microbial species. In this work, solid electrodes with controlled potentials were placed under mats to study the electron transfer processes between the electrode and the microbial mat. The phototrophic microbial mat was harvested from Hot Lake, a hypersaline, epsomitic lake located near Oroville (Washington, USA). We operated two reactors: graphite electrodes were polarized at potentials of -700 mVAg/AgCl [cathodic (CAT) mat system] and +300 mVAg/AgCl [anodic (AN) mat system] and the electron transfer rates between the electrode and mat were monitored. We observed a diel cycle of electron transfer rates for both AN and CAT mat systems. Interestingly, the CAT mats generated the highest reducing current at the same time points that the AN mats showed the highest oxidizing current. To characterize the physicochemical factors influencing electron transfer processes, we measured depth profiles of dissolved oxygen (DO) and sulfide in the mats using microelectrodes. We further demonstrated that the mat-to-electrode and electrode-to-mat electron transfer rates were light- and temperature-dependent. Using nuclear magnetic resonance (NMR) imaging, we determined that the electrode potential regulated the diffusivity and porosity of the microbial mats. Both porosity and diffusivity were higher in the CAT mats than in the AN mats. We also used NMR spectroscopy for high-resolution quantitative metabolite analysis and found that the CAT mats had significantly higher concentrations of osmoprotectants such as betaine and trehalose. Subsequently, we performed amplicon sequencing across the V4 region of the 16S rRNA gene of incubated mats to understand the impact of electrode potential on microbial community structure. These data suggested that variation in the electrochemical conditions under which mats were generated significantly impacted the relative abundances of mat members and mat metabolism.

Expression, Purification, and Characterization of a Recombinant Methionine Adenosyltransferase pDS16 in Pichia pastoris

Methionine adenosyltransferase (MAT, EC2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (SAM) using L-methionine and adenosine triphosphate (ATP) as substrates. The mutant MAT pDS16 was obtained through DNA shuffling previously in our lab. Overexpression of pDS16 in Pichia pastoris led to about 65 % increase of MAT activity and SAM accumulation, compared with the strain overexpressing Saccharomyces cerevisiae MAT gene SAM2. Different strategies were tested to facilitate the expression and purification of pDS16. However, addition of the hexahistidine tag to pDS16 was shown to decrease the enzyme activity, and the yeast α-factor signal sequence could not effectivley direct the secretion of pDS16. The intracellular pDS16 was purified by a simple two-step procedure combining an ion exchange and hydrophobic interaction chromatography. Protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 93%, with the specific activity of 1.828 U/mg. Two-dimensional electrophoresis revealed pI of ∼5.5. The purified enzyme followed Michaelis kinetics with a Km of 1.72 and 0.85 mM, and Vmax of 1.54 and 1.15 μmol/min/mg for ATP and L-methionine, respectively. pDS16 exhibited optimal activity at pH 8.5 and 45 °C with the requirement of divalent cation Mg(2+) and was slightly stimulated by the monovalent cation K(+). It showed an improved thermostability, about 50% of the enzyme activity was retained even after preincubation at 50 °C for 2 h.

GAP Promoter Library for Fine-Tuning of Gene Expression in Pichia pastoris

A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast Pichia pastoris is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in P. pastoris, we created a functional promoter library through mutagenesis of the constitutive GAP promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (β-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and S-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris.

Distinctive fungal and bacterial communities are associated with mats formed by ectomycorrhizal fungi

The distinct rhizomorphic mats formed by ectomycorrhizal Piloderma fungi are common features of the organic soil horizons of coniferous forests of the Pacific Northwest. These mats have been found to cover 25–40% of the forest floor in some Douglas-fir stands, and are associated with physical and biochemical properties that distinguish them from the surrounding non-mat soils. In this study, we examined the fungal and bacterial communities associated with Piloderma mat and non-mat soils. Each mat and non-mat area was repeatedly sampled at four times throughout the year. Characterization of the mat activity and community was achieved using a combination of N-acetylglucosaminidase (NAGase) enzyme assays, and molecular analysis of fungal and bacterial communities using T-RFLP profiles, clone libraries, and quantitative PCR. Piloderma mats had consistently greater NAGase activity across all dates, although the magnitude of the difference varied by season. Furthermore, we found distinct fungal and bacterial communities associated with the Piloderma mats, yet the size of the microbial populations differed little between the mat and non-mat soils. Significant temporal variation was seen in the NAGase activity and in the sizes of the fungal and bacterial populations, but the community composition remained stable through time. Our results demonstrate the presence of two distinct microbial communities occupying the forest floor of Douglas-fir stands, whose populations and activities fluctuate seasonally but with little change in composition, which appears to be related to the physiochemical nature of mat and non-mat habitats.

Enzymatic activity of methionine adenosyltransferase variants identified in patients with persistent hypermethioninemia

Methionine adenosyltransferases (MAT's) are central enzymes in living organisms that have been conserved with a high degree of homology among species. In the liver, MAT I and III, tetrameric and dimeric isoforms of the same catalytic subunit encoded by the gene MAT1A, account for the predominant portion of total body synthesis of S-adenosylmethionine (SAM), a versatile sulfonium ion-containing molecule involved in a variety of vital metabolic reactions and in the control of hepatocyte proliferation and differentiation. During the past 15years 28 MAT1A mutations have been described in patients with elevated plasma methionines, total homocysteines at most only moderately elevated, and normal levels of tyrosine and other aminoacids. In this study we describe functional analyses that determine the MAT and tripolyphosphatase (PPPase) activities of 18 MAT1A variants, six of them novel, and none of them previously assayed for activity. With the exception of G69S and Y92H, all recombinant proteins showed impairment (usually severe) of MAT activity. Tripolyphosphate (PPPi) hydrolysis was decreased only in some mutant proteins but, when it was decreased MAT activity was always also impaired.

Microbial Species Richness and Metabolic Activities in Hypersaline Microbial Mats: Insight into Biosignature Formation Through Lithification

Microbial mats in the hypersaline lake of Salt Pan, Eleuthera, Bahamas, display a gradient of lithification along a transect from the center to the shore of the lake. These mats exist under similar geochemical conditions, with light quantity and quality as the sole major environmental difference. Therefore, we hypothesized that the microbial community may be driving the differences in lithification and, by extension, mineral biosignature formation. The lithifying and non-lithifying mat communities were compared (via 16S rRNA gene sequencing, 485 and 464 sequences, respectively) over both temporal and spatial scales. Seven bacterial groups dominated in all the microbial mat libraries: bacteriodetes, alphaproteobacteria, deltaproetobacteria, chloroflexi, spirochaetes, cyanobacteria, and planctomycetes. The mat communities were all significantly different over space, time, and lithification state. Species richness is significantly higher in the non-lithifying mats, potentially due to differences in mat structure and activity. This increased richness may impact lithification and, hence, biosignature production.

DNA shuffling of methionine adenosyltransferase gene leads to improved S-adenosyl- l-methionine production in Pichia pastoris

S-Adenosyl- l-methionine (SAM) is an important molecule for normal cell function and survival. To enhance methionine adenosyltransferase (MAT, EC2.5.1.6) activity and thus SAM production in recombinant Pichia pastoris, MAT genes from Escherichia coli, Saccharmyces cerevisiae, and Streptomyces spectabilis were recombined by DNA shuffling. The shuffled genes were transformed into P. pastoris GS115 to construct recombinant strains and screened for high MAT activity and enhanced SAM production mutants. In the two best recombinant strains, the MAT activities were respectively 201% and 65% higher than the recombinant strains containing the starting MAT genes, and the SAM concentration increased by 103% and 65% respectively. The analysis on the deduced sequences of five representative MAT variants showed that the K18R mutation probably resulted in the increased activity of the best MAT, which may provide new insights into structure-enzyme activity relationship of MAT and might shed light on the further rational evolution of MAT. Finally, a 6.14 g l −1 of SAM production was reached in a 500 l bioreactor with the best recombinant strain GS115/DS56, which showed a favorable foreground in industrial scale production of SAM.

Characterization of a methionine adenosyltransferase over-expressing strain in the trypanosomatid Leishmania donovani

Methionine adenosyltransferase (MAT: EC 2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (AdoMet) in two sequential steps, AdoMet formation and subsequent tripolyphosphate (PPPi) cleavage, induced by AdoMet. In pursuit of a better understanding of the biological function of the enzyme, the MAT gene was cloned into vector PX63NEO to induce episomal overexpression in leishmania parasites. Neomycin-selected clones originated a strain of such overexpressing parasites that accumulated more than 3-fold AdoMet than mock-transfected cells and showed over ten times the wild type MAT activity, concurring with a significant accumulation of the MAT protein during the early logarithmic phase and MAT transcripts throughout the growth cycle. The rate of AdoMet efflux, practically nil in the control promastigotes, was exceptionally high in the MAT-overexpressing parasites, whilst growth in this strain was comparable to development in control cells, i.e., it was not affected by deleterious hypermethylation. Moreover, the modified strain was 10-fold more resistant to sinefungin, a S-adenosylmethionine-like antibiotic, than control cells. The effects of overexpression on polyamine metabolism and transport were likewise studied.

Mutational analysis of methionine adenosyltransferase from Leishmania donovani.

The methionine adenosyltransferase (MAT; EC 2.5.1.6) mediated synthesis of S-adenosylmethionine (AdoMet) is a two-step process, consisting of the formation of AdoMet and the subsequent cleavage of the tripolyphosphate (PPPi) molecule, a reaction induced, in turn, by AdoMet. The fact that the two activities--AdoMet synthesis and tripolyphosphate hydrolysis--can be measured separately is particularly useful when the site-directed mutagenesis approach is used to determine the functional role of the amino acid residues involved in each. This report describes the mutational analysis of the amino acids involved in both the ATP and L-methionine binding sites of Leishmania donovani MAT (GenBank accession number AF179714) the aetiological agent of visceral leishmaniasis. Site-directed mutagenesis was used to substitute neutral residues for the basic amino acid (Lys168, Lys256, Lys276, Lys280 and His17), acidic residues (Asp19, Asp121, Asp166, Asp249, Asp277 and Asp288) and Phe241 involved in AdoMet synthesis and PPPi hydrolysis. With the exception of D116N, none of these mutants was able to synthesize AdoMet at a significant rate, although H17A, H17N, K256A, K280A, D19N, D121N, D166N, D249N and D282N showed measurable tripolyphosphatase activity. Finally, the C-terminus domain of L. donovani MAT was truncated at three points (F382Stop, D375Stop, F368Stop), deleting a 3(10) one-turn helix motif in all three cases. Whilst none of the truncated proteins conserved MAT activity, they were able to hydrolyse PPPi, albeit at a lower rate than the wild-type enzyme. A fourth protein with an internal deletion (E376DeltaF382) in the C-terminal domain conserved high tripolyphosphatase activity, which was not, however, induced by 50 microM AdoMet.

Interactions between heavy metals and clay matrix in fluid catalytic cracking catalysts

The interactions between heavy metals (vanadium and nickel) and kaolin had been investigated by X-ray diffraction and MAT test. In the process of phase transformation of kaolin containing 1–5% vanadium, vanadium-mullite phase emerged surprisingly at 660–700 °C. The unit cell size of the mullite was found to increase with increment of vanadium content. As compared with hydro-kaolin, acid-modified kaolin (AMK) and caustic-modified kaolin (CMK) reacted with vanadium to form much more mullite; CMK could even be reacted with nickel to form a rather stable NiAl 10O 16 compound, and this compound protected zeolite in fluid catalytic cracking catalyst effectively. The catalyst containing AMK had greater MAT activity retention than that of the contrastive catalyst with hydro-kaolin in the identical level of heavy metal contamination.

High-performance liquid chromatography determination of methionine adenosyltransferase activity using catechol- O-methyltransferase-coupled fluorometric detection

A nonradioactive, sensitive, rapid, and specific method for the determination of methionine adenosyltransferase activity has been established. In this method, the methyl group of S-adenosyl- l-methionine was enzymatically transferred to esculetin with the aid of catechol- O-methyltransferase and then the resulting scopoletin was extracted with n-hexane:ethyl acetate (7:3, v/v) and measured by high-performance liquid chromatography with Si 60 column and fluorometric detection with excitation and emission wavelengths at 347 and 415 nm, respectively. The detection limit for scopoletin was about 100 fmol. Using this method to determine MAT activity in HL-60 cells required only about 2.5 μg of protein and the incubation time needed for enzymatic reaction is less than 30 min. The HPLC analysis procedure took only 5 min per sample. The kinetic study showed that MAT in HL-60 cells exhibited negative cooperativity with a Hill coefficient of 0.5. The values of K m and V max were 6.1 ± 0.3 μM and 135.4 ± 1.5 nmol AdoMet formed/mg protein/h, respectively.

Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling

S-adenosylmethionine synthetase (ATP: l-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10 −2 s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the “R1” residue reduced the k cat only for the labeled D107R1 form (7-fold). The K m value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K m for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by ∼10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the viscosity dependence of the rate of AdoMet formation. The results suggest that the motion of the loop may be an intrinsic property of the protein and not be strictly ligand modulated.