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10 p.-4 fig., Focal adhesion kinase (FAK; also known as PTK2) was discovered three decades ago and is now recognised as a key player in the regulation of cell–matrix adhesion and mesenchymal cell migration. Although it is essential during development, FAK also drives invasive cancer progression and metastasis. On a structural level, the basic building blocks of FAK have been described for some time. However, a picture of how FAK integrates into larger assemblies in various cellular environments, including one of its main cellular locations, the focal adhesion (FA) complex, is only beginning to emerge. Nano-resolution data from cellular studies, as well as atomic structures from reconstituted systems, have provided first insights, but also point to challenges that remain for obtaining a full structural understanding of how FAK is integrated in the FA complex and the structural changes occurring at different stages of FA maturation. In this Review, we discuss the known structural features of FAK, the interactions with its partners within the FA environment on the cell membrane and propose how its initial assembly in nascent FAs might change during FA maturation under force., D.L. is funded by the Ministerio de Ciencia, Innovación y Universidades for the Spanish State Research Agency Retos (grant RTI2018-099318-B-I00), and from the Ministerio de Ciencia e Innovación for the Generation of Knowledge (grant PID2021-127058NB-I00), both co-funded by the European Regional Development Fund (FEDER).

13 págs., 6 figs., 3 tabs., Nucleoside 2′-deoxyribosyltransferases (NDTs) catalyze the cleavage of glycosidic bonds of 2′-deoxynucleosides and the following transfer of the 2′-deoxyribose moiety to acceptor nucleobases. Here, we report the crystal structures and biochemical properties of the first tetrameric NDTs: the type I NDT from the mesophilic bacterium Enterococcus faecalis V583 (EfPDT) and the type II NDT from the bacterium Desulfotalea psychrophila (DpNDT), the first psychrophilic NDT. This novel structural and biochemical data permitted an exhaustive comparative analysis aimed to shed light into the basis of the high global stability of the psychrophilic DpNDT, which has a higher melting temperature than EfPDT (58.5 °C versus 54.4 °C) or other mesophilic NDTs. DpNDT possesses a combination of unusual structural motifs not present neither in EfPDT nor any other NDT that most probably contribute to its global stability, in particular, a large aliphatic isoleucine-leucine-valine (ILV) bundle accompanied by a vicinal disulfide bridge and also an intersubunit disulfide bridge, the first described for an NDT. The functional and structural features of DpNDT do not fit the standard features of psychrophilic enzymes, which lead us to consider the implication of (sub)cellular levels together with the protein level in the adaptation of enzymatic activity to low temperatures., This work was supported by grants CTQ2009-11543 (IDLM), PID2020-117025RB-I00 (JF-L) and AGL2017-84614-C2-2-R (JMM) from the Spanish Ministry of Science and Innovation, S2009/PPQ-1752 (IDLM) from Comunidad Autonoma de Madrid and XSAN192006 (IDLM) from the Santander Foundation. This research was funded in part by National Institutes of Health grant (GM115586 to AJ). The use of SBC beamlines at the Advanced Photon Source is supported by the U.S. Department of Energy (DOE) Office of Science and operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357 (AJ).

21 p.-9 fig.-2 tab., Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions., I.A. acknowledges support from the José Castillejo Mobility Grant issued by the Spanish Ministry of Education, Culture and Sports. R.D.R. acknowledges funding from the Fellowships for Excellence program sponsored by the Werner-Siemens Foundation and the University of Basel.H.S. acknowledges support by the Swiss National Science Foundation(grants 205320_166164 and 185544 (NCCR TransCure)). M.C.F., A.B., J.C., and C.S. were supported by a Cancer Research UK Programme Grant (C157/ A24837) to M.C.F. F.G. acknowledges funding by the Klaus Tschira Foundation, by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy – 2082/1 –390761711, and the state of Baden-Württemberg through bwHPC and the DFG through grant INST 35/11341 FUGG.S.d.B. thanks the Carl Zeiss Foundation for financial support. D.L. acknowledges support from the Spanish Ministry of Economy, Industry and Competitiveness for the Retos Grant BFU2016-77665-R and the Ministry of Science, Innovation and Universities for the Spanish State Research Agency Retos Grant RTI2018-099318-B-I00,both co-funded by the European Regional Development Fund (FEDER) and from an intramural grant from the Spanish National Research Council(CSIC, Ref: 201820I124). O.L. is funded by SAF2017-82632-P grant from the Spanish Ministry of Science, Innovation and Universities (MCIU/AEI), cofunded by the European Regional Development Fund (ERDF), by the National Institute of Health Carlos III and by projects Y2018/BIO4747 and P2018/NMT4443 from the Autonomous Region of Madrid and co-funded by the European Social Fund and the European Regional Development Fund. C.F.R. is funded by a BES-2015-071348 PhD fellowship by the Spanish Ministry of Science, Innovation and Universities (MCIU/AEI).

Statistics from structural genomics initiatives reveal that around 50–55% of the expressed, non-membrane proteins cannot be purified and therefore structurally characterized due to solubility problems, which emphasized protein solubility as one of the most serious concerns in structural biology projects. Lactobacillus plantarum CECT 748 produces an aggregation-prone glycosidase (LpBgl) that we crystallized previously. However, this result could not be reproduced due to protein instability and therefore further high-resolution structural analyses of LpBgl were impeded. The obtained crystals of LpBgl diffracted up to 2.48 Å resolution and permitted to solve the structure of the enzyme. Analysis of the active site revealed a pocket for phosphate-binding with an uncommon architecture, where a phosphate molecule is tightly bound suggesting the recognition of 6-phosphoryl sugars. In agreement with this observation, we showed that LpBgl exhibited 6-phospho-β-glucosidase activity. Combination of structural and mass spectrometry results revealed the formation of dimethyl arsenic adducts on the solvent exposed cysteine residues Cys211 and Cys292. Remarkably, the double mutant Cys211Ser/Cys292Ser resulted stable in solution at high concentrations indicating that the marginal solubility of LpBgl can be ascribed specifically to these two cysteine residues. The 2.30 Å crystal structure of this double mutant showed no disorder around the newly incorporated serine residues and also loop rearrangements within the phosphate-binding site. Notably, LpBgl could be prepared at high yield by proteolytic digestion of the fusion protein LSLt-LpBgl, which raises important questions about potential hysteretic processes upon its initial production as an enzyme fused to a solubility enhancer., We thank the ESRF (Grenoble, France) for provision of synchrotron radiation facilities (BM16 and ID23-2). Financial support from the Ministerio de Economía y Competitividad (BFU2010-17929/BMC) (to J.M.M.) and AGL2014-52911-R (to R.M.).

The N-acetylglucosaminidase NagZ of Pseudomonas aeruginosa catalyzes the first cytoplasmic step in recycling of muropeptides, cell-wall-derived natural products. This reaction regulates gene expression for the β-lactam resistance enzyme, β-lactamase. The enzyme catalyzes hydrolysis of N-acetyl-β-d-glucosamine-(1→4)-1,6-anhydro-N-acetyl-β-d-muramyl-peptide (1) to N-acetyl-β-d-glucosamine (2) and 1,6-anhydro-N-acetyl-β-d-muramyl-peptide (3). The structural and functional aspects of catalysis by NagZ were investigated by a total of seven X-ray structures, three computational models based on the X-ray structures, molecular-dynamics simulations and mutagenesis. The structural insights came from the unbound state and complexes of NagZ with the substrate, products and a mimetic of the transient oxocarbenium species, which were prepared by synthesis. The mechanism involves a histidine as acid/base catalyst, which is unique for glycosidases. The turnover process utilizes covalent modification of D244, requiring two transition-state species and is regulated by coordination with a zinc ion. The analysis provides a seamless continuum for the catalytic cycle, incorporating large motions by four loops that surround the active site.

Endogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni 2+-NTA matrices becoming a potential contaminant for recombinant, target His-tagged proteins. Purified recombinant, untagged GPDH (rGPDH) converted galactitol into tagatose, and d-tagatose-6-phosphate into galactitol-1-phosphate, in a Zn 2+- and NAD(H)-dependent manner and readily crystallized what has permitted to solve its crystal structure. In contrast, N-terminally His-tagged GPDH was marginally stable and readily aggregated. The structure of rGPDH revealed metal-binding sites characteristic from the medium-chain dehydrogenase/reductase protein superfamily which may explain its ability to interact with immobilized metals. The structure also provides clues on the harmful effects of the N-terminal His-tag. Structured summary of protein interactions: GPDH and GPDH bind by molecular sieving (View interaction) GPDH and GPDH bind by x-ray crystallography (View interaction) GPDH and GPDH bind by cosedimentation in solution (View interaction). © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved., JMM and RM thank the Ministerio de Ciencia e Innovación for their research grantsBFU2010-17929, CSD2006-00015 and AGL2011-22745, CSD2007-00063 FUN-C-FOOD and Comunidad de Madrid S2009/AGR-1469, respectively.

In this work, we analyzed at high resolution the sugar-binding mode of the recombinant N-terminal ricin-B domain of the hemolytic protein LSLa (LSL150) from the mushroom Laetiporus sulphureus and also provide functional in vitro evidence suggesting that, together with its putative receptor-binding role, this module may also increase the solubility of its membrane pore-forming partner. We first demonstrate that recombinant LSL150 behaves as an autonomous folding unit and an active lectin. We have determined its crystal structure at 1.47 Å resolution and also that of the [LSL150:(lactose)β, γ)] binary complex at 1.67 Å resolution. This complex reveals two lactose molecules bound to the β and γ sites of LSL150, respectively. Isothermal titration calorimetry indicates that LSL150 binds two lactoses in solution with highly different affinities. Also, we test the working hypothesis that LSL150 exhibits in vivo properties typical of solubility tags. With this aim, we have fused an engineered version of LSL150 (LSLt) to the N-terminal end of various recombinant proteins. All the designed LSL150-tagged fusion proteins were successfully produced at high yield, and furthermore, the target proteins were purified by a straightforward affinity procedure on agarose-based matrices due to the excellent properties of LSL150 as an affinity tag. An optimized protocol for target protein purification was devised, which involved removal of the LSL150 tag through in-column cleavage of the fusion proteins with His6-tagged TEV endoprotease. These results permitted to set up a novel, lectin-based system for production and purification of recombinant proteins in E. coli cells with attractive biotechnological applications., Financial support from the Ministerio de Ciencia e Innovación (BFU2010-17929/BMC; BFU2009-10052) and the Factoría de Cristalización (Consolider-Ingenio-2007), and CIBER de Enfermedades Respiratorias (CIBERES), an initiative of Instituto de Salud Carlos III, is greatly appreciated.

In recent years, the exquisite stereoselectivity and high efficiency of carbohydrate-processing enzymes have been exploited for many biotechnological applications, including flavor enhancement in foods. In particular, much attention has been focused on the use of b-glucosidases for the enzymatic hydrolysis of flavorless glycoconjugates present in juices and wine beverages for the release aroma volatiles. With the aim to analyze a novel glycosidase with potential applications food industry we have produced and structurally characterized the Bgl glycosidase from the food lactic acid bacterium Lactobacillus plantarum. For that purpose, we have cloned and heterologously expressed the bgl gene (lp_3629) in Escherichia coli. The recombinant protein containing an amino terminal His6 tag (Bgl) has been produced in a soluble form. Purified recombinant enzyme shows galactosidase activity against 4-nitrophenyl b-D-galactopyranoside but not glucosidase activity. Analytical size-exclusion gel filtration chromatography reveals that Bgl behaves in solution as a mixture of monomeric and a high-molecular weight assembly. Purified Bgl has been crystallized by the hanging-drop vapor-diffusion method at 18 C. Diffraction data have been collected at ESRF to a resolution of 2.4 Å. The crystals belong to the space group C2 with unit-cell parameters a = 196.7, b = 191.7, c = 105.9, b = 102.7 . The structure refinement is in progress, JMM thanks the Ministerio de Educación y Ciencia for a research grant (BFU2007-67404/BMC) and ‘‘Factoría de Cristalización” Consolider- Ingenio 2010 in support of his research and the ESRF (Grenoble, France) for provision of synchrotron radiation facilities. This work was also supported by Grants AGL2008-01052 and CSD2007-00063 FUN-C-FOOD (MICINN), RM2008-00002 (INIA), and S-0505/AGR/000153 ALIBIRD (CAM). J.A. Curiel is a recipient of a FPI predoctoral fellowship from the MICINN.

Bacteria grow and divide without loss of cellular integrity. This accomplishment is notable, as a key component of their cell envelope is a surrounding glycopeptide polymer. In Gram-negative bacteria this polymer—the peptidoglycan—grows by the difference between concurrent synthesis and degradation. The regulation of the enzymatic ensemble for these activities is poorly understood. We report herein the structural basis for the control of one such enzyme, the lytic transglycosylase MltF of Pseudomonas aeruginosa. Its structure comprises two modules: an ABC-transporter-like regulatory module and a catalytic module. Occupancy of the regulatory module by peptidoglycan-derived muropeptides effects a dramatic and long-distance (40 Å) conformational change, occurring over the entire protein structure, to open its active site for catalysis. This discovery of the molecular basis for the allosteric control of MltF catalysis is foundational to further study of MltF within the complex enzymatic orchestration of the dynamic peptidoglycan.

Design of generic methods aimed at the oriented attachment of proteins at the interfacial environment of magnetic nanoparticles currently represents an active field of research. With this in mind, we have prepared and characterized agarose-coated maghemite nanoparticles to set up a platform for the attachment of recombinant proteins fused to the β-trefoil lectin domain LSL, a small protein that combines fusion tag properties with agarose-binding capacity. Analysis of the agarose-coated nanoparticles by dynamic light scattering, Fourier transform infrared spectroscopy, and thermogravimetric studies shows that decoupling particle formation from agarose coating provides better results in terms of coating efficiency and particle size distribution. LSL interacts with these agarose-coated nanoparticles exclusively through the recognition of the sugars of the polymer, forming highly stable complexes, which in turn can be dissociated ad hoc with the competing sugar lactose. Characterization of the complexes formed with the fusion proteins LSL-EGFP (LSL-tagged enhanced green fluorescent protein from Aquorea Victoria) and LSL-BTL2 (LSL-tagged lipase from Geobacillus thermocatenolatus) provided evidence supporting a topologically oriented binding of these molecules to the interface of the agarose-coated nanoparticles. This is consistent with the marked polarity of the β-trefoil structure where the sugar-binding sites and the N- and C-terminus ends are at opposed sides. In summary, LSL displays topological and functional features expected from a generic molecular adaptor for the oriented attachment of proteins at the interface of agarose-coated nanoparticles., This work was partially supported by grant No. MAT2014-52069-R from the Spanish Ministry of Economy and Competitiveness (M.P.M.), by C-KBBE/3293 project from the European Commission (J.M.G.) and by grant BFU2010-17929 from the Spanish Ministry of Science and Innovation (J.M.M.). I.A. and Y.L. hold a predoctoral FPU fellowship from the Spanish Ministry of Economy and Competitiveness. A.G.R.-E. holds a predoctoral fellowship from a CSIC–CITMA collaborative project (ICMM, 2011-2014).

The hydrolase fold is one of the most versatile structures in the protein realm according to the diversity of sequences adopting such a three-dimensional architecture. In the present study, we clarified the crystal structure of the carboxylesterase Cest-2923 from the lactic acid bacterium Lactobacillus plantarum WCFS1 refined to 2.1 Å resolution, determined its main biochemical characteristics and also carried out an analysis of its associative behaviour in solution. We found that the versatility of a canonical α/β hydrolase fold, the basic framework of the crystal structure of Cest-2923, also extends to its oligomeric behaviour in solution. Thus, we discovered that Cest-2923 exhibits a pH-dependent pleomorphic behaviour in solution involving monomers, canonical dimers and tetramers. Although, at neutral pH, the system is mainly shifted to dimeric species, under acidic conditions, tetrameric species predominate. Despite these tetramers resulting from the association of canonical dimers, as is commonly found in many other carboxylesterases from the hormone-sensitive lipase family, they can be defined as 'noncanonical' because they represent a different association mode. We identified this same type of tetramer in the closest relative of Cest-2923 that has been structurally characterized: the sugar hydrolase YeeB from Lactococcus lactis. The observed associative behaviour is consistent with the different crystallographic results for Cest-2923 from structural genomics consortia. Finally, the presence of sulfate or acetate molecules (depending on the crystal form analysed) in the close vicinity of the nucleophile Ser116 allows us to identify interactions with the putative oxyanion hole and deduce the existence of hydrolytic activity within Cest-2923 crystals. Structured digital abstract Cest-2923 and Cest-2923 bind by x-ray crystallography (1, 2)Cest-2923 and Cest-2923 bind by cosedimentation in solution (1, 2) Database The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 4BZW for Cest-2923 from native crystals not soaked with substrates (P6322 space group); 4C01 for Cest-2923 from crystals soaked with phenyl acetate (C2 space group); 4BZZ for Cest-2923 from crystals soaked with isopropenyl acetate (P622 space group). The analysis of the carboxylesterase Cest-2923 from the lactic acid bacterium Lactobacillus plantarum WCFS1 reveals that the intrinsically structural versatility of a canonical α/β-hydrolase fold also extends to its oligomeric behaviour in solution. Hence, we found that Cest-2923, exhibits a complex, pH-dependent pleomorphic behaviour involving monomers, canonical dimers and noncanonical tetramers. © 2013 FEBS., Financial support received from the Ministerio de Economıa y Competitividad (BFU2010-17929/BMC), as well as from Factorıa de Cristalizacion (Consolider-Ingenio-2007) (J.M.M.), AGL2011-22745 (R.M.) and FUN-C-FOOD Consolider 25506 (R.M.), is greatly appreciated. Y.A., M.E.-T. and I.A. are recipients of fellowships: CSICCITMA, JAE Predoc (CSIC) and FPU (MEC), respectively.

A highly stable lipase from Geobacillus thermocatenolatus (BTL2) and the enhanced green fluorescent protein from Aquorea victoria (EGFP) were recombinantly produced N-terminally tagged to the lectin domain of the hemolytic pore-forming toxin LSLa from the mushroom Laetiporus sulphureus. Such a domain (LSL 150), recently described as a novel fusion tag, is based on a β-trefoil scaffold with two operative binding sites for galactose or galactose-containing derivatives. The fusion proteins herein analyzed have enabled us to characterize the binding mode of LSL 150 to polymeric and solid substrates such as agarose beads. The lectin-fusion proteins are able to be quantitatively bound to both cross-linked and non-cross-linked agarose matrixes in a very rapid manner, resulting in a surprisingly dynamic protein distribution inside the porous beads that evolves from heterogeneous to homogeneous along the postimmobilization time. Such dynamic distribution can be related to the reversible nature of the LSL 150-agarose interaction. Furthermore, this latter interaction is temperature dependent since it is 4-fold stronger when the immobilization takes place at 25 °C than when it does at 4 °C. The strongest lectin-agarose interaction is also quite stable under a survey of different conditions such as high temperatures (up to 60 °C) or high organic solvent concentrations (up to 60% of acetonitrile). Notably, the use of cross-linked agarose would endow the system with more robustness due to its better mechanical properties compared to the noncross-linked one. The stability of the LSL 150-agarose interaction would prevent protein leaching during the operation process unless high pH media are used. In summary, we believe that the LSL 150 lectin domain exhibits interesting structural features as an immobilization domain that makes it suitable to reversibly immobilize industrially relevant enzymes in very simple carriers as agarose.

Q88Y25_Lacpl is an esterase produced by the lactic acid bacterium Lactobacillus plantarum WCFS1 that shows amino-acid sequence similarity to carboxylesterases from the hormone-sensitive lipase family, in particular the AFEST esterase from the archaeon Archaeoglobus fulgidus and the hyperthermophilic esterase EstEI isolated from a metagenomic library. N-terminally His6-tagged Q88Y25_Lacpl has been overexpressed in Escherichia coli BL21 (DE3) cells, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. Mass spectrometry was used to determine the purity and homogeneity of the enzyme. Crystals of His6-tagged Q88Y25_Lacpl were prepared in a solution containing 2.8 M sodium acetate trihydrate pH 7.0. X-ray diffraction data were collected to 2.24 A ¿ resolution on beamline ID29 at the ESRF. The apparent crystal point group was 422; however, initial global analysis of the intensity statistics (data processed with high symmetry in space group I422) and subsequent tests on data processed with low symmetry (space group I4) showed that the crystals were almost perfectly merohedrally twinned. Most probably, the true space group is I4, with unit-cell parameters a = 169.05, b = 169.05, c = 183.62 A, JMM and RM thank the Ministerio de Educación y Ciencia for research grants (BFU2010-17929/BMC, AGL2008-01052 and AGL2011-22745; DGCYT), ‘Factoría de Cristalización’ (CSD2006-00015) and FUN-C-FOOD (CSD2007-00063) Consolider-Ingenio 2010 and ALIBIRD S2009/AGR-1469 (CAM) and RM2008-00002 (INIA) for support of their research.

5 páginas, 4 figuras, 1 tabla -- PAGS nros. 549-553, NicX from Pseudomonas putida KT2440 is an Fe2+-dependent dioxygenase that is involved in the aerobic degradation of nicotinic acid. The enzyme converts 2,5-dihydroxypyridine to N-formylmaleamic acid when overexpressed in Escherichia coli. Biophysical characterization of NicX by analytical gel-filtration chromatography revealed that it behaves as an oligomeric assembly in solution, with an apparent molecular weight that is consistent with a hexameric species. NicX was crystallized by the hanging-drop vapour-diffusion method at 291 K. Diffraction data were collected to a resolution of 2.0 Å at the ESRF. The crystals most probably belong to the orthorhombic space group C222 or C2221. The estimated Matthews coefficient was 2.4 Å3 Da-1, corresponding to 50% solvent content, which is consistent with the presence of three protein molecules in the asymmetric unit. Analysis of the crystal data together with chromatographic results supports NicX being a hexameric assembly composed of two cyclic trimers. Currently, crystallization of recombinant selenomethionine-containing NicX is in progress, JMM thanks the Ministerio de Educación y Ciencia for a research grant (BFU2007-67404/BMC), `Factoría de Cristalización' Consolider-Ingenio 2010 for support of his research and the ESRF (Grenoble, France) for provision of synchrotron-radiation facilities. ED and JLG acknowledge the Ministerio de Ciencia e Innovación for grants GEN2006-27750-C5-3-E, BIO2006-05957 and CSD2007-00005 and the Comunidad Autónoma de Madrid for grant P-AMB-259-0505. IA is the recipient of an FPU predoctoral fellowship from the Ministerio de Educación. JIJ was the recipient of an I3P predoctoral fellowship from the Consejo Superior de Investigaciones Científicas

Tannase is an enzyme with important biotechnological applications in the food industry. Previous studies have identified the tannase encoding gene in Lactobacillus plantarum and also have reported the description of the purification of recombinant L. plantarum tannase through a protocol involving several chromatographic steps. Here, we describe the high-yield production of pure recombinant tannase (17 mg/L) by a one-step affinity procedure. The purified recombinant tannase exhibits optimal activity at pH 7 and 40 C. Addition of Ca2þ to the reaction mixture greatly increased tannase activity. The enzymatic activity of tannase was assayed against 18 simple phenolic acid esters. Only esters derived from gallic acid and protocatechuic acid were hydrolyzed. In addition, tannase activity was also assayed against the tannins tannic acid, gallocatechin gallate, and epigallocatechin gallate. Despite L. plantarum tannase representing a novel family of tannases, which shows no significant similarity to tannases from fungal sources, both families of enzymes shared similar substrate specificity range. The physicochemical characteristics exhibited by L. plantarum recombinant tannase make it an adequate alternative to the currently used fungal tannases, This work was supported by grants AGL2005-00470, AGL2008-01052, BFU2007-67404/BMC, Consolider INGENIO 2010 CSD2007-00063 FUN-C-FOOD, and Consolider INGENIO 2007 Factoria de Crystalización (CICYT), RM2008-00002 (INIA), and S-0505/AGR/000153 (CAM). H. Rodríguez and J. A. Curiel were recipients of predoctoral fellowships from the I3P-CSIC Program and FPI-MEC, respectively.

© 2015 Bentham Science Publishers. The ability to resist the effect of a wide range of antibiotics makes methicillin-resistant Staphylococcus aureus (MRSA) a leading global human pathogen. A key determinant of resistance to β-lactam antibiotics in this organism is penicillin-binding protein 2a (PBP2a), an enzyme that catalyzes the crosslinking reaction between two adjacent peptide stems during the peptidoglycan biosynthesis. The recently published crystal structure of the complex of PBP2a with ceftaroline, a cephalosporin antibiotic that shows efficacy against MRSA, has revealed the allosteric site at 60-A distance from the transpeptidase domain. Binding of ceftaroline to the allosteric site of PBP2a triggers conformational changes that lead to the opening of the active site from a closed conformation, where a second molecule of ceftaroline binds to give inhibition of the enzyme. The discovery of allostery in MRSA remains the only known example of such regulation of cellwall biosynthesis and represents a new paradigm in fighting MRSA. This review summarizes the present knowledge of the allosteric mechanism, the conformational changes allowing PBP2a catalysis and the means by which some clinical strains have acquired resistance to ceftaroline by disrupting the allosteric mechanism.