Helena Santos

New University of Lisbon, Lisboa, Lisbon, Portugal

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Publications (158)523.56 Total impact

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    ABSTRACT: Halophilic and halotolerant microorganisms adapted to thrive in hot environments accumulate compatible solutes that usually have a negative charge either associated with a carboxylic group or a phosphodiester unit. Mannosylglycerate (MG) has been detected in several members of (hyper)thermophilic bacteria and archaea, in which it responds primarily to osmotic stress. The outstanding ability of MG to stabilize protein structure in vitro as well as in vivo has been convincingly demonstrated. These findings led to an increasingly supported link between MG and microbial adaptation to high temperature. However, the accumulation of MG in many red algae has been known for a long time, and the peculiar distribution of MG in such distant lineages was intriguing. Knowledge on the biosynthetic machinery together with the rapid expansion of genome databases allowed for structural and phylogenetic analyses and provided insight into the distribution of MG. The two pathways for MG synthesis have distinct evolutionary histories and physiological roles: in red algae MG is synthesised exclusively via the single-step pathway and most probably is unrelated with stress protection. In contrast, the two-step pathway is strongly associated with osmoadaptation in (hyper)thermophilic prokaryotes. The phylogenetic analysis of the two-step pathway also reveals a second cluster composed of fungi and mesophilic bacteria, but MG has not been demonstrated in members of this cluster; we propose that the synthase is part of a more complex pathway directed at the synthesis of yet unknown molecules containing the mannosyl-glyceryl unit.
    Extremophiles : life under extreme conditions. 08/2014;
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    ABSTRACT: The nutritionally versatile soil bacterium Acinetobacter baylyi ADP1 copes with salt stress by the accumulation of compatible solutes, a strategy that is widespread in nature. This bacterium synthesizes the sugar alcohol mannitol de novo in response to osmotic stress. In a previous study, we identified MtlD, a mannitol-1-phosphate dehydrogenase, which is essential for mannitol biosynthesis and which catalyzes the first step in mannitol biosynthesis, the reduction of fructose-6-phosphate (F-6-P) to the intermediate mannitol-1-phosphate (Mtl-1-P). Until now, the identity of the second enzyme, the phosphatase that catalyzes the dephosphorylation of Mtl-1-P to mannitol, was elusive. Here we show that MtlD has a unique sequence among known mannitol-1-phosphate dehydrogenases with a haloacid dehalogenases (HAD)-like phosphatase domain at the N-terminus. This domain is indeed shown to have a phosphatase activity. Phosphatase activity is strictly Mg(2+) dependent. NMR analysis revealed that purified MtlD catalyzes not only reduction of F-6-P but also dephosphorylation of Mtl-1-P. MtlD of A. baylyi is the first bifunctional enzyme of mannitol biosynthesis that combines Mtl-1-P dehydrogenase and phosphatase activities in a single polypeptide chain. Bioinformatic analysis revealed that the bifunctional enzyme is wide-spread among Acinetobacter strains but only rarely present in other phylogenetic tribes.
    Environmental Microbiology 05/2014; · 6.24 Impact Factor
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    ABSTRACT: Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supra-optimal temperature or salinity. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at a supra-optimal temperature and NaCl concentration. DIP was a suitable replacement for MG during heat stress, but substitution of MG by DIP and aspartate led to a less efficient growth under osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG is as effective as DIP to protect cells against heat, despite the finding that DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.
    Applied and Environmental Microbiology 05/2014; · 3.95 Impact Factor
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    ABSTRACT: Corynebacterium glutamicum wild type produces a mixture of lactic, succinic and acetic acid from glucose under oxygen deprivation. We investigated the effect of CO2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose and, subsequently, organic acid production by non-growing cells was studied under anaerobic conditions. The presence of CO2 caused an up to 3-fold increase in the succinate yield (1 mol per mol of glucose), and about 2-fold increase in acetate, both at the expense of L-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO2 was estimated by using (13)C-labelled glucose and (13)C-NMR analysis of the labelling patterns in end-products. The flux analysis showed that 97% succinate was produced via the reductive part of the tricarboxylic acid cycle, the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (∼5%) regardless of the presence or absence of CO2. Moreover, there was a significant channelling of carbon to storage compounds (glycogen, trehalose), and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo-NMR and the stoichiometry (H(+):organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation.
    Applied and Environmental Microbiology 03/2014; · 3.95 Impact Factor
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    ABSTRACT: Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.
    Frontiers in Microbiology 01/2014; 5:168. · 3.90 Impact Factor
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    ABSTRACT: Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65 Å resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane α-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed.
    Nature Communications 01/2014; 5:4169. · 10.74 Impact Factor
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    ABSTRACT: Details of the beamline, detector and overall Wilson B in the article by Nogly et al. (2012, Acta Cryst. F68, 1113-1115) are corrected.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2013; 69(Pt 11):1313. · 0.55 Impact Factor
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    ABSTRACT: The first structure of a bacterial α-phosphoglucomutase with an overall fold similar to eukaryotic phosphomannomutases is reported. Unlike most α-phosphoglucomutases within the α-D-phosphohexomutase superfamily, it belongs to subclass IIb of the haloacid dehalogenase superfamily (HADSF). It catalyzes the reversible conversion of α-glucose 1-phosphate to glucose 6-phosphate. The crystal structure of α-phosphoglucomutase from Lactococcus lactis (APGM) was determined at 1.5 Å resolution and contains a sulfate and a glycerol bound at the enzyme active site that partially mimic the substrate. A dimeric form of APGM is present in the crystal and in solution, an arrangement that may be functionally relevant. The catalytic mechanism of APGM and its strict specificity towards α-glucose 1-phosphate are discussed.
    Acta Crystallographica Section D Biological Crystallography 10/2013; 69(Pt 10):2008-2016. · 12.67 Impact Factor
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    ABSTRACT: BACKGROUND: Protein aggregation in the brain is a central hallmark in many neurodegenerative diseases. In Parkinson's disease, α-synuclein (α-Syn) is the major component of the intraneuronal inclusions found in the brains of patients. Current therapeutics is merely symptomatic, and there is a pressing need for developing novel therapies. Previously we showed that mannosylglycerate (MG), a compatible solute typical of marine microorganisms thriving in hot environments, is highly effective in protecting a variety of model proteins against thermal denaturation and aggregation in vitro. METHODS: Saccharomyces cerevisiae cells expressing eGFP-tagged α-Syn, were further engineered to synthesize MG. The number of cells with fluorescent foci was assessed by fluorescence microscopy. Fluorescence spectroscopy and transmission electron microscopy were used to monitor fibril formation in vitro. RESULTS: We observed a 3.3-fold reduction in the number of cells with α-Syn foci and mild attenuation of α-Syn-induced toxicity. Accordingly, sucrose gradient analysis confirmed a clear reduction in the size-range of α-Syn species in the cells. MG did not affect the expression levels of α-Syn or its degradation rate. Moreover, MG did not induce molecular chaperones (Hsp104, Hsp70 and Hsp40), suggesting the implication of other mechanisms for α-Syn stabilization. MG also inhibited α-Syn fibrillation in vitro. CONCLUSIONS: MG acts as a chemical chaperone and the stabilization mechanism involves direct solute/protein interactions. GENERAL SIGNIFICANCE: This is the first demonstration of the anti-aggregating ability of MG in the intracellular milieu. The work shows that MG is a good candidate to inspire the development of new drugs for protein-misfolding diseases.
    Biochimica et Biophysica Acta 04/2013; · 4.66 Impact Factor
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    ABSTRACT: Double quantum and triple quantum filtered (23)Na nuclear magnetic resonance techniques were used to characterise in detail the isotropic and anisotropic binding and dynamics of intra- and extracellular Na(+) in different cellular systems, in the absence and presence of Li(+). The kinetics of Li(+) influx by different cell types was evaluated. At steady state, astrocytes accumulated more Li(+) than red blood cells (RBCs), while a higher intracellular Li(+) concentration was found in chromaffin than in SH-SY5Y cells. Anisotropic and isotropic motions were detected for extracellular Na(+) in all cellular systems studied. Isotropic intracellular Na(+) motions were observed in all types of cells, while anisotropic Na(+) motions in the intracellular compartment were only detected in RBCs. (23)Na triple quantum signal efficiency for intracellular Na(+) was SH-SY5Y > chromaffin > RBCs, while the reverse order was observed for the extracellular ions. (23)Na double quantum signal efficiency for intracellular Na(+) was non-zero only in RBCs, and for extracellular Na(+) the order RBCs > chromaffin > SH-SY5Y cells was observed. Li(+) loading generally decreased intracellular Na(+) isotropic movements in the cells, except for astrocytes incubated with a low Li(+) concentration and increased anisotropic intracellular Na(+) movements in RBCs. Li(+) effects on the extracellular signals were more complex, reflecting Li(+)/Na(+) competition for isotropic and anisotropic binding sites at the extracellular surface of cell membranes and also at the surface of the gel used for cell immobilisation. These results are relevant and contribute to the interpretation of the in vivo pharmacokinetics and sites of Li(+) action.
    Biophysics of Structure and Mechanism 04/2013; · 2.44 Impact Factor
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    ABSTRACT: The lactic acid bacteria (LAB) are a functionally related group of low-GC Gram-positive bacteria known essentially for their roles in bioprocessing of foods and animal feeds. Due to extensive industrial use and enormous economical value, LAB have been intensively studied and a large body of comprehensive data on their metabolism and genetics was generated throughout the years. This knowledge has been instrumental in the implementation of successful applications in the food industry, such as the selection of robust starter cultures with desired phenotypic traits. The advent of genomics, functional genomics and high-throughput experimentation combined with powerful computational tools currently allows for a systems level understanding of these food industry workhorses. The technological developments in the last decade have provided the foundation for the use of LAB in applications beyond the classic food fermentations. Here we discuss recent metabolic engineering strategies to improve particular cellular traits of LABand to design LAB cell factories for the bioproduction of added value chemicals.
    Biotechnology advances 04/2013; · 8.25 Impact Factor
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    ABSTRACT: The nutritionally versatile and naturally competent soil bacterium Acinetobacter baylyi copes with salt stress by the accumulation of compatible solutes. NMR analyses revealed that cells amassed glutamate and the rather unusual sugar alcohol mannitol upon an increase of the external NaCl concentration. To unravel the path of mannitol biosynthesis, the genome was inspected for genes potentially involved in its biosynthesis. A gene encoding a potential mannitol-1-phosphate dehydrogenase (mtlD) was identified in the genome of A. baylyi. Expression of mtlD was highly induced at high salinity. mtlD was overexpressed and the purified protein indeed produced mannitol-1-phosphate from fructose-6-phosphate. The enzyme preferred NADPH over NADH and the specific activity of fructose-6-phosphate reduction with NADPH was 130 U mg(-1) . Enzymatic activity was strictly salt-dependent. Deletion of mtlD resulted in a complete loss of salt-dependent mannitol biosynthesis. We provide clear evidence that osmo-induced synthesis of the compatible solute mannitol is by a two-step pathway and that the mannitol-1-phosphate dehydrogenase mediating the first step of this pathway is regulated by salinity on the transcriptional as well as on the activity level.
    Environmental Microbiology 01/2013; · 6.24 Impact Factor
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    ABSTRACT: The effect of pH on the glucose metabolism of non-growing cells of L. lactis MG1363 was studied by in vivo NMR in the range 4.8 to 6.5. Immediate pH effects on glucose transporters and/or enzyme activities were distinguished from transcriptional/translational effects by using cells grown at the optimal pH of 6.5 or pre-adjusted to low pH by growth at 5.1. In cells grown at pH 5.1, glucose metabolism proceeds at a rate 35% higher than in non-adjusted cells at the same pH. Besides the upregulation of stress-related genes (such as dnaK and groEL), cells adjusted to low pH overexpressed H(+)-ATPase subunits as well as glycolytic genes. At sub-optimal pHs, the total intracellular pool of lactic acid reached approximately 500 mM in cells grown at optimal pH and about 700 mM in cells grown at pH 5.1. These high levels, together with good pH homeostasis (internal pH always above 6), imply intracellular accumulation of the ionized form of lactic acid (lactate anion), and the concomitant export of the equivalent protons. The average number, n, of protons exported with each lactate anion was determined directly from the kinetics of accumulation of intra- and extracellular lactic acid as monitored online by (13)C-NMR. In cells non-adjusted to low pH, n varies between 2 and 1 during glucose consumption, suggesting an inhibitory effect of intracellular lactate on proton export. We confirmed that extracellular lactate did not affect the lactate: proton stoichiometry. In adjusted cells, n was lower and varied less, indicating a different mix of lactic acid exporters less affected by the high level of intracellular lactate. A qualitative model for pH effects and acid stress adaptation is proposed on the basis of these results.
    PLoS ONE 01/2013; 8(7):e68470. · 3.53 Impact Factor
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    ABSTRACT: The accumulation of organic solutes was investigated in the thermophilic bacteria Persephonella marina and Marinitoga piezophila, two representatives of the deepest lineages in the domain Bacteria. These organisms grow optimally at around 70 °C in medium containing 3 % NaCl. A new disaccharide, accumulating in Persephonella marina, was identified as α(1-6)glucosyl-α(1-2)glucosylglycerate (GGG), by nuclear magnetic resonance. This identification was validated by comparison with the spectra of the compound obtained by chemical synthesis. Besides GGG, the solute pool of Persephonella marina comprised β-glutamate, di-myo-inositol-1,3'-phosphate and 2-O-α-glucosylglycerate. In contrast, amino acids such as α-glutamate, proline and alanine were the dominant components of the solute pool of Marinitoga piezophila and sugar derivatives were absent. The ability of GGG to protect protein structure against heat denaturation was assessed using model proteins. A genomic search for the biosynthetic pathways of known ionic solutes in Aquificales and Thermotogales shows the inability of this analysis to predict the nature of compatible solutes and underlines the need for efficient cultivation techniques.
    Extremophiles 11/2012; · 2.20 Impact Factor
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    ABSTRACT: α-Phosphoglucomutase (α-PGM) is an enzyme that is essential for the growth of Lactococcus lactis. The enzyme links bacterial anabolism with sugar utilization through glycolysis by catalyzing the reversible interconversion of glucose 6-phosphate and α-glucose 1-phosphate. The gene encoding α-PGM was cloned and overexpressed in L. lactis. The purified protein was functionally active and was crystallized with ammonium sulfate as a precipitant using vapour-diffusion and seeding techniques. Optimized crystals diffracted to 1.5 Å resolution at a synchrotron source.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 09/2012; 68(Pt 9):1113-5. · 0.55 Impact Factor
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    ABSTRACT: Mannosylglycerate is a compatible solute typical of thermophilic marine microorganisms that has a remarkable ability to protect proteins from thermal denaturation. This ionic solute appears to be a universal stabilizing agent, but the extent of protection depends on the specific protein examined. To understand how mannosylglycerate confers protection, we have been studying its influence on the internal motions of a hyperstable staphylococcal nuclease (SNase). Previously, we found a correlation between the magnitude of protein stabilization and the restriction of fast backbone motions. We now report the effect of mannosylglycerate on the fast motions of side-chains and on the slower unfolding motions of the protein. Side-chain motions were assessed by (13)CH(3) relaxation measurements and model-free analysis while slower unfolding motions were probed by H/D exchange measurements at increasing concentrations of urea. Side-chain motions were little affected by the presence of different concentrations of mannosylglycerate or even by the presence of urea (0.25M), and show no correlation with changes in the thermodynamic stability of SNase. Native hydrogen exchange experiments showed that, contrary to reports on other stabilizing solutes, mannosylglycerate restricts local motions in addition to the global motions of the protein. The protein unfolding/folding pathway remained undisturbed in the presence of mannosylglycerate but the solute showed a specific effect on the local motions of β-sheet residues. This work reinforces the link between solute-induced stabilization and restriction of protein motions at different timescales, and shows that the solute preferentially affects specific structural elements of SNase.
    Protein Science 05/2012; 21(8):1126-37. · 2.74 Impact Factor
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    Helena Santos
    FEBS Letters. 03/2012; 586(5):476–478.
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    ABSTRACT: The synthesis of di-myo-inositol phosphate (DIP), a common compatible solute in hyperthermophiles, involves the consecutive actions of inositol-1-phosphate cytidylyltransferase (IPCT) and di-myo-inositol phosphate phosphate synthase (DIPPS). In most cases, both activities are present in a single gene product, but separate genes are also found in a few organisms. Genes for IPCT and DIPPS were found in the genomes of 33 organisms, all with thermophilic/hyperthermophilic lifestyles. Phylogeny of IPCT/DIPPS revealed an incongruent topology with 16S RNA phylogeny, thus suggesting horizontal gene transfer. The phylogenetic tree of the DIPPS domain was rooted by using phosphatidylinositol phosphate synthase sequences as out-group. The root locates at the separation of genomes with fused and split genes. We propose that the gene encoding DIPPS was recruited from the biosynthesis of phosphatidylinositol. The last DIP-synthesizing ancestor harboured separated genes for IPCT and DIPPS and this architecture was maintained in a crenarchaeal lineage, and transferred by horizontal gene transfer to hyperthermophilic marine Thermotoga species. It is plausible that the driving force for the assembly of those two genes in the early ancestor is related to the acquired advantage of DIP producers to cope with high temperature. This work corroborates the view that Archaea were the first hyperthermophilic organisms.
    Environmental Microbiology 03/2012; 14(3):691-701. · 6.24 Impact Factor
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    ABSTRACT: Background / Purpose: To understand the molecular mechanisms underlying protein stabilization by compatible solutes, we studied the effect of different solutes on the dynamics of a staphylococcal nuclease variant (SNase) at different time scales.NMR spin relaxation was used to measure the effect of mannosylglycerate (a strong stabilizer), urea, KCl, and glycerol on the sub-nanosecond motions of backbone amide protons and side-chain methyl groups. Main conclusion: We found a clear inverse relationship of backbone motions and changes in the melting temperature (T m) induced by different solutes, (i.e., decreased backbone dynamics correlated with increased T m), but not for side chain motions. NH exchange rate measurements were used to probe the slower local and global motions of the protein. We found that mannosylglycerate (MG) restrains global motions in a relatively uniform way but local motions were unexpectedly found to be preferentially restricted in the beta-sheets of SNase.These results go some way to explain the observation that stabilisation can be strongly dependent on the specific solute/protein pair under study.
    Biochemical Society 2011 - Dynamics Within and Between Proteins meeting; 10/2011
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    ABSTRACT: Mannosyl-3-phosphoglycerate phosphatase (MpgP) is a key mediator in the physiological response to thermal and osmotic stresses, catalyzing the hydrolysis of mannosyl-3-phosphoglycerate (MPG) to the final product, α-mannosylglycerate. MpgP is a metal-dependent haloalcanoic acid dehalogenase-like (HAD-like) phosphatase, preserving the catalytic motifs I-IV of the HAD core domain, and classified as a Cof-type MPGP (HAD-IIB-MPGP family; SCOP [117505]) on the basis of its C2B cap insertion module. Herein, the crystallographic structures of Thermus thermophilus HB27 MpgP in its apo form and in complex with substrates, substrate analogues, and inhibitors are reported. Two distinct enzyme conformations, open and closed, are catalytically relevant. Apo-MpgP is primarily found in the open state, while holo-MpgP, in complex with the reaction products, is found in the closed state. Enzyme activation entails a structural rearrangement of motifs I and IV with concomitant binding of the cocatalytic Mg(2+) ion. The closure motion of the C2B domain is subsequently triggered by the anchoring of the phosphoryl group to the cocatalytic metal center, and by Arg167 fixing the mannosyl moiety inside the catalytic pocket. The results led to the proposal that in T. thermophilus HB27 MpgP the phosphoryl transfer employs a concerted D(N)S(N) mechanism with assistance of proton transfer from the general acid Asp8, forming a short-lived PO(3)(-) intermediate that is attacked by a nucleophilic water molecule. These results provide new insights into a possible continuum of phosphoryl transfer mechanisms, ranging between those purely associative and dissociative, as well as a picture of the main mechanistic aspects of phosphoryl monoester transfer catalysis, common to other members of the HAD superfamily.
    Biochemistry 09/2011; 50(44):9551-67. · 3.38 Impact Factor

Publication Stats

2k Citations
523.56 Total Impact Points

Institutions

  • 1995–2014
    • New University of Lisbon
      • • Institute of Chemical and Biological Technology (ITQB)
      • • Faculty of Sciences and Technology
      Lisboa, Lisbon, Portugal
  • 2006–2013
    • Goethe-Universität Frankfurt am Main
      • Institut für Molekulare Biowissenschaften
      Frankfurt am Main, Hesse, Germany
    • Georgia Institute of Technology
      • Department of Biomedical Engineering
      Atlanta, GA, United States
  • 2010
    • University of Groningen
      • Department of Molecular Genetics
      Groningen, Province of Groningen, Netherlands
  • 2007–2010
    • Technical University of Lisbon
      • • Centro de Química Estrutural (CQE)
      • • Departamento de Engenharia Química (DEQ)
      Lisbon, Lisbon, Portugal
  • 1998–2010
    • University of Coimbra
      • • Centro de Neurociências e Biologia Celular (CNC)
      • • Departamento de Zoologia
      • • Department of Chemistry
      Coimbra, Distrito de Coimbra, Portugal
  • 2009
    • Kyoto University
      • Department of Synthetic Chemistry and Biological Chemistry
      Kioto, Kyōto, Japan
  • 2008
    • Spanish National Research Council
      • Biological Research Centre
      Madrid, Madrid, Spain
  • 2003–2008
    • Universität Konstanz
      • Department of Biology
      Constance, Baden-Württemberg, Germany
  • 1982–2005
    • Instituto Técnico y Cultural
      Santa Clara de Portugal, Michoacán, Mexico
  • 1996–2001
    • Instituto de Biologia Experimental e Tecnológica IBET
      • Animal Cell Technology Unit
      Portugal
  • 1998–2000
    • University of Southampton
      • Faculty of Natural and Environmental Sciences
      Southampton, ENG, United Kingdom
  • 1999
    • MIT Portugal
      Porto Salvo, Lisbon, Portugal
  • 1990
    • University of Georgia
      • Department of Microbiology
      Атина, Georgia, United States