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1.  Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi 
The variable C-terminal tail of manganese peroxidases, a group of enzymes involved in lignin degradation, is implicated in their catalytic and stability properties, as shown by new crystal structures, molecular-simulation and directed-mutagenesis data. Based on this structural–functional evaluation, short and long/extralong manganese peroxidase subfamilies have been accepted; the latter are characterized by exceptional stability, while it is shown for the first time that the former are able to oxidize other substrates at the same site where manganese(II) is oxidized.
The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+ oxidation by the internal propionate, but prevents the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+ binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+ and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.
PMCID: PMC4257621  PMID: 25478843
Ceriporiopsis subvermispora; manganese peroxidase; C-terminal tail
2.  Ligninolytic peroxidase genes in the oyster mushroom genome: heterologous expression, molecular structure, catalytic and stability properties, and lignin-degrading ability 
The genome of Pleurotus ostreatus, an important edible mushroom and a model ligninolytic organism of interest in lignocellulose biorefineries due to its ability to delignify agricultural wastes, was sequenced with the purpose of identifying and characterizing the enzymes responsible for lignin degradation.
Heterologous expression of the class II peroxidase genes, followed by kinetic studies, enabled their functional classification. The resulting inventory revealed the absence of lignin peroxidases (LiPs) and the presence of three versatile peroxidases (VPs) and six manganese peroxidases (MnPs), the crystal structures of two of them (VP1 and MnP4) were solved at 1.0 to 1.1 Å showing significant structural differences. Gene expansion supports the importance of both peroxidase types in the white-rot lifestyle of this fungus. Using a lignin model dimer and synthetic lignin, we showed that VP is able to degrade lignin. Moreover, the dual Mn-mediated and Mn-independent activity of P. ostreatus MnPs justifies their inclusion in a new peroxidase subfamily. The availability of the whole POD repertoire enabled investigation, at a biochemical level, of the existence of duplicated genes. Differences between isoenzymes are not limited to their kinetic constants. Surprising differences in their activity T50 and residual activity at both acidic and alkaline pH were observed. Directed mutagenesis and spectroscopic/structural information were combined to explain the catalytic and stability properties of the most interesting isoenzymes, and their evolutionary history was analyzed in the context of over 200 basidiomycete peroxidase sequences.
The analysis of the P. ostreatus genome shows a lignin-degrading system where the role generally played by LiP has been assumed by VP. Moreover, it enabled the first characterization of the complete set of peroxidase isoenzymes in a basidiomycete, revealing strong differences in stability properties and providing enzymes of biotechnological interest.
PMCID: PMC3902061  PMID: 24387130
Genome; Pleurotus ostreatus; Ligninolytic peroxidase genes; Heterologous expression; Crystal structure; Catalytic properties; Thermal stability; pH stability; Gene duplication; Peroxidase evolution
3.  Effect of Transcriptional Activators SoxS, RobA, and RamA on Expression of Multidrug Efflux Pump AcrAB-TolC in Enterobacter cloacae 
Antimicrobial Agents and Chemotherapy  2012;56(12):6256-6266.
Control of membrane permeability is a key step in regulating the intracellular concentration of antibiotics. Efflux pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae. The AcrAB-TolC efflux pump is involved in multidrug resistance in Enterobacter cloacae. However, the underlying mechanism that regulates the system in this microorganism remains unknown. In Escherichia coli, the transcription of acrAB is upregulated under global stress conditions by proteins such as MarA, SoxS, and Rob. In the present study, two clinical isolates of E. cloacae, EcDC64 (a multidrug-resistant strain overexpressing the AcrAB-TolC efflux pump) and Jc194 (a strain with a basal AcrAB-TolC expression level), were used to determine whether similar global stress responses operate in E. cloacae and also to establish the molecular mechanisms underlying this response. A decrease in susceptibility to erythromycin, tetracycline, telithromycin, ciprofloxacin, and chloramphenicol was observed in clinical isolate Jc194 and, to a lesser extent in EcDC64, in the presence of salicylate, decanoate, tetracycline, and paraquat. Increased expression of the acrAB promoter in the presence of the above-described conditions was observed by flow cytometry and reverse transcription-PCR, by using a reporter fusion protein (green fluorescent protein). The expression level of the AcrAB promoter decreased in E. cloacae EcDC64 derivates deficient in SoxS, RobA, and RamA. Accordingly, the expression level of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing SoxS, RobA, and RamA. Overall, the data showed that SoxS, RobA, and RamA regulators were associated with the upregulation of acrAB, thus conferring antimicrobial resistance as well as a stress response in E. cloacae. In summary, the regulatory proteins SoxS, RobA, and RamA were cloned and sequenced for the first time in this species. The involvement of these proteins in conferring antimicrobial resistance through upregulation of acrAB was demonstrated in E. cloacae.
PMCID: PMC3497196  PMID: 23006750
4.  Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue 
Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2.
At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O2 for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state.
Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.
PMCID: PMC3483288  PMID: 22913590
Hypoxia; Horse; AT-MSC; BM-MSC; Characterisation
5.  Design, Synthesis and Crystal Structures of 6-Alkylidene-2’-Substituted Penicillanic Acid Sulfones as Potent Inhibitors of Acinetobacter baumannii OXA-24 Carbapenemase 
Journal of the American Chemical Society  2010;132(38):13320-13331.
Class D β-lactamases represent a growing and diverse class of penicillin inactivating enzymes that are usually resistant to commercial β-lactamase inhibitors. As many such enzymes are found in multi-drug resistant (MDR) Acinetobacter baumannii and Pseudomonas aeruginosa, novel β-lactamase inhibitors are urgently needed. Five unique 6-alkylidene-2’-substituted penicillanic acid sulfones (1, 2, 3, 4, and 5) were synthesized and tested against OXA-24, a clinically important β-lactamase that inactivates carbapenems and found in A. baumannii. Based upon the roles Tyr112 and Met223 play in the OXA-24 β-lactamase, we also engineered two variants (Tyr112Ala and Tyr112Ala,Met223Ala) to test the hypothesis that the hydrophobic tunnel formed by these residues influences inhibitor recognition. IC50 values, against OXA-24, and two OXA-24 β-lactamase variants ranged from 10 ± 1 (4 vs. WT) to 338 ± 20 nM (5 vs. Tyr112Ala, Met223Ala). Compound 4 possessed the lowest Ki (500 ± 80 nM vs. WT) and 1 possessed the highest inactivation efficiency (kinact/Ki = 0.21 ± 0.02 μM-1s-1). Electrospray ionization mass spectrometry revealed a single covalent adduct, suggesting the formation of an acyl-enzyme intermediate. X-ray structures of OXA-24 complexed to four inhibitors (2.0-2.6 Å) reveal there is formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanion hole. These data provide the first structural evidence that 6-alkylidene-2’-substituted penicillin sulfones are effective mechanism-based inactivators of class D β-lactamases. Their unique chemistry makes them developmental candidates. Mechanisms for class D hydrolysis and inhibition are discussed, and a pathway for the evolution of the BlaR1 sensor of Staphylococcus aureus to the class D β-lactamases is proposed.
PMCID: PMC3393087  PMID: 20822105
OXA carbapenemase; β-lactamase inhibitor; penicillin
6.  New Biofuel Integrating Glycerol into Its Composition Through the Use of Covalent Immobilized Pig Pancreatic Lipase 
By using 1,3-specific Pig Pancreatic lipase (EC or PPL), covalently immobilized on AlPO4/Sepiolite support as biocatalyst, a new second-generation biodiesel was obtained in the transesterification reaction of sunflower oil with ethanol and other alcohols of low molecular weight. The resulting biofuel is composed of fatty acid ethyl esters and monoglycerides (FAEE/MG) blended in a molar relation 2/1. This novel product, which integrates glycerol as monoacylglycerols (MG) into the biofuel composition, has similar physicochemical properties compared to those of conventional biodiesel and also avoids the removal step of this by-product. The biocatalyst was found to be strongly fixed to the inorganic support (75%). Nevertheless, the efficiency of the immobilized enzyme was reduced to half (49.1%) compared to that of the free PPL. The immobilized enzyme showed a remarkable stability as well as a great reusability (more than 40 successive reuses) without a significant loss of its initial catalytic activity. Immobilized and free enzymes exhibited different reaction mechanisms, according to the different results in the Arrhenius parameters (Ln A and Ea). However, the use of supported PPL was found to be very suitable for the repetitive production of biofuel due to its facile recyclability from the reaction mixture.
PMCID: PMC3431847  PMID: 22949849
biofuels; sunflower oil transesterification; covalent immobilization; pig pancreatic lipase (PPL); Sepiolite; amorphous AlPO4, monoglyceride
7.  The Binding of Triclosan to SmeT, the Repressor of the Multidrug Efflux Pump SmeDEF, Induces Antibiotic Resistance in Stenotrophomonas maltophilia 
PLoS Pathogens  2011;7(6):e1002103.
The wide utilization of biocides poses a concern on the impact of these compounds on natural bacterial populations. Furthermore, it has been demonstrated that biocides can select, at least in laboratory experiments, antibiotic resistant bacteria. This situation has raised concerns, not just on scientists and clinicians, but also on regulatory agencies, which are demanding studies on the impact that the utilization of biocides may have on the development on resistance and consequently on the treatment of infectious diseases and on human health. In the present article, we explored the possibility that the widely used biocide triclosan might induce antibiotic resistance using as a model the opportunistic pathogen Stenotrophomonas maltophilia. Biochemical, functional and structural studies were performed, focusing on SmeDEF, the most relevant antibiotic- and triclosan-removing multidrug efflux pump of S. maltophilia. Expression of smeDEF is regulated by the repressor SmeT. Triclosan released SmeT from its operator and induces the expression of smeDEF, thus reducing the susceptibility of S. maltophilia to antibiotics in the presence of the biocide. The structure of SmeT bound to triclosan is described. Two molecules of triclosan were found to bind to one subunit of the SmeT homodimer. The binding of the biocide stabilizes the N terminal domain of both subunits in a conformation unable to bind DNA. To our knowledge this is the first crystal structure obtained for a transcriptional regulator bound to triclosan. This work provides the molecular basis for understanding the mechanisms allowing the induction of phenotypic resistance to antibiotics by triclosan.
Author Summary
The wide utilization of biocides for different purposes, including toothpastes, soaps, house-hold compounds surfaces' disinfectants and even their use as additives of different materials (from textiles to concrete used in germ-free buildings) to avoid their colonization by microorganisms, poses a concern on the impact of these compounds on natural bacterial populations. Furthermore, it has been demonstrated that such biocides can select, at least in laboratory experiments, bacteria resistant to antibiotics. This situation has raised concerns on the impact that the utilization of biocides may have on the development on resistance and consequently on the treatment of infectious diseases. In the present article we study whether biocides can induce phenotypic resistance to antibiotics, a process that would be barely detectable unless purposely searched out. In the article, we present functional, biochemical and structural data showing that the widely used biocide triclosan induces antibiotic resistance, mediated by the binding of the biocide to SmeT, the transcriptional regulator of the expression of the Stenotrophomonas maltophilia multidrug efflux pump SmeDEF, which can extrude an ample range of antibiotics. Our study provides an unambiguous link between the presence of this biocide and the increased efflux of antibiotics by the opportunistic pathogen S. maltophilia.
PMCID: PMC3128119  PMID: 21738470
8.  Crystallization and preliminary crystallographic studies of human kallikrein 7, a serine protease of the multigene kallikrein family 
The cloning, expression, purification and crystallization of recombinant human kallikrein 7, directly synthesized in the active form in E. coli, is described. Diffraction data were collected to 2.8 Å resolution from native crystals.
Human kallikreins are a group of serine proteases of high sequence homology whose genes are grouped as a single cluster at chromosome 19. Although the physiological roles of kallikreins are generally still unknown, members of the kallikrein family have been clearly implicated in pathological situations such as cancer and psoriasis. Human kallikrein 7 (hK7) has been shown to be involved in pathological keratinization, psoriasis and ovarian cancer. In order to gain insight into the molecular structure of this protein, hK7 was crystallized after recombinant production in its folded and active form using a periplasmic secretion vector in Escherichia coli. The crystals belonged to the rhombohedral space group H32 and diffracted to 2.8 Å. The phase problem was solved by molecular replacement using the mouse kallikrein-related protein neuropsin. Completion of the model and structure refinement are under way.
PMCID: PMC2335154  PMID: 17671364
kallikrein 7; serine proteases

Results 1-8 (8)