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Phytase is used in poultry diets to hydrolyze and release of phytate-bound phosphorus. Immobilization on nanomaterials optimizes enzyme's thermal stability and reusability. This study aimed to immobilize the recombinant phytase from Yersinia intermedia on the surface of amino-multi-walled carbon nanotubes (amino-MWCNTs) by physical adsorption. For this, zeta potential measurement, FTIR spectroscopic analysis, scanning electron microscope (SEM), kinetic as well as thermodynamic parameters were used to characterize immobilized phytase on amino-MWCNTs. According to results, the optimum temperature of the immobilized phytase increased from 50 to 70 °C and also thermal and pH stability improved considerably. Moreover, immobilization led to an increase in the value of K m and k cat from 0.13 to 0.33 mM and 2220 to 2776 s -1 , respectively. In addition, the changes in activation energy of thermal inactivation (ΔE # a (D) ), the free energy of thermal inactivation (ΔG # D ) and the enthalpy of thermal inactivation (ΔH # D ) for immobilized phytase increased by +11.05, +24.7 and +11.4 kj/mole, respectively, while the value of the change in the entropy of thermal inactivation (ΔS # D ) decreased by - 0.04 kj/mole.K. Overall, our results showed that adsorption immobilization of phytase on amino-MWCNTs increases thermal, pH and storage stability as well as some of kinetic parameters., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Being able to recombine more than two genes with four or more crossover points in a sequence independent manner is still a challenge in protein engineering and limits our capabilities in tailoring enzymes for industrial applications. By computational analysis employing multiple sequence alignments and homology modeling, five fragments of six phytase genes (sequence identities 31-64 %) were identified and efficiently recombined through phosphorothioate-based cloning using the PTRec method. By combinatorial recombination, functional phytase chimeras containing fragments of up to four phytases were obtained. Two variants (PTRec 74 and PTRec 77) with up to 32 % improved residual activity (90 °C, 60 min) and retained specific activities of > 1100 U/mg were identified. Both variants are composed of fragments from the phytases of Citrobacter braakii, Hafnia alvei and Yersinia mollaretii. They exhibit sequence identities of ≤ 80 % to their parental enzymes, highlighting the great potential of DNA recombination strategies to generate new enzymes with low sequences identities that offer opportunities for property right claims., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We identified a glucosyltransferase (YGT) and an ADP-ribosyltransferase (YART) in Yersinia mollaretii , highly related to glucosylating toxins from Clostridium difficile , the cause of antibiotics-associated enterocolitis. Both Yersinia toxins consist of an amino-terminal enzyme domain, an autoprotease domain activated by inositol hexakisphosphate, and a carboxyl-terminal translocation domain. YGT N -acetylglucosaminylates Rab5 and Rab31 at Thr 52 and Thr 36 , respectively, thereby inactivating the Rab proteins. YART ADP-ribosylates Rab5 and Rab31 at Gln 79 and Gln 64 , respectively. This activates Rab proteins by inhibiting GTP hydrolysis. We determined the crystal structure of the glycosyltransferase domain of YGT (YGT G ) in the presence and absence of UDP at 1.9- and 3.4-Å resolution, respectively. Thereby, we identified a previously unknown potassium ion-binding site, which explains potassium ion-dependent enhanced glycosyltransferase activity in clostridial and related toxins. Our findings exhibit a novel type of inverse regulation of Rab proteins by toxins and provide new insights into the structure-function relationship of glycosyltransferase toxins., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Enzyme immobilization is extensively studied to improve enzyme properties in catalysis and analytical applications. Here, we introduce a simple and versatile enzyme immobilization platform based on adhesion-promoting peptides, namely Matter-tags. Matter-tags immobilize enzymes in an oriented way as a dense monolayer. The immobilization platform was established with three adhesion-promoting peptides; Cecropin A (CecA), liquid chromatography peak I (LCI), and Tachystatin A2 (TA2), that were genetically fused to enhanced green fluorescent protein and to two industrially important enzymes: a phytase (from Yersinia mollaretii) and a cellulase (CelA2 from a metagenomic library). Here, we report a universal and simple Matter-tag-based immobilization platform for enzymes on various materials including polymers (polystyrene, polypropylene, and polyethylene terephthalate), metals (stainless steel and gold), and silicon-based materials (silicon wafer). The Matter-tag-based enzyme immobilization is performed at ambient temperature within minutes (<10 min) in an aqueous solution harboring the phytase or cellulase by immersing the targeted material. The peptide LCI was identified as universal adhesion promoter; LCI immobilized both enzymes on all investigated materials. The attachment of phytase-LCI onto gold was characterized with surface plasmon resonance spectroscopy obtaining a dissociation constant value (K D ) of 2.9·10 -8  M and a maximal surface coverage of 504 ng/cm²., (© 2019 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

A nuclease from Yersinia enterocolitica subsp. palearctica (Nucyep) is a newly found thermostable nonspecific nuclease. The heat-resisting ability of this nuclease would be extremely useful in biological research or pharmaceutical production. However, the application of this nuclease is limited because of its poor yield. This research aimed to improve Nucyep productivity by producing a novel genetically engineered Escherichia coli and optimizing the production procedures. After 4 h of induction by lactose, the new genetically engineered E. coli can express a substantial amount of Nucyep in the form of inclusion bodies. The yield was approximately 0.3 g of inclusion bodies in 1 g of bacterial pellets. The inclusion bodies were extracted by sonication and solubilized in an 8 M urea buffer. Protein renaturation was successfully achieved by dilution method. Pure enzyme was obtained after subjecting the protein solution to anion exchange. The Nucyep showed its nonspecific and heat resistant properties as previously reported (Boissinot et  al. 2016). Through a quantification method, its activity was determined to be 1.3 × 10 6 Kunitz units (K.U.)/mg. These results can serve as a reference for increasing Nucyep production., (© FEMS 2019.)

Yersinia Protein Tyrosine Phosphatase (YopH) is the most efficient enzyme among all known PTPases and relies on its catalytic loop movements for substrate binding and catalysis. Fluorescence, NMR, and UV resonance Raman (UVRR) techniques have been used to study the thermodynamic and dynamic properties of the loop motions. In this study, a computational approach based on the pathway refinement methods nudged elastic band (NEB) and harmonic Fourier beads (HFB) has been developed to provide structural interpretations for the experimentally observed kinetic processes. In this approach, the minimum potential energy pathways for the loop open/closure conformational changes were determined by NEB using a one-dimensional global coordinate. Two dimensional data analyses of the NEB results were performed as an efficient method to qualitatively evaluate the energetics of transitions along several specific physical coordinates. The free energy barriers for these transitions were then determined more precisely using the HFB method. Kinetic parameters were estimated from the energy barriers using transition state theory and compared against experimentally determined kinetic parameters. When the calculated energy barriers are calibrated by a simple "scaling factor", as have been done in our previous vibrational frequency calculations to explain the ligand frequency shift upon its binding to protein, it is possible to make structural interpretations of several observed enzyme dynamic rates. For example, the nanosecond kinetics observed by fluorescence anisotropy may be assigned to the translational motion of the catalytic loop and microsecond kinetics observed in fluorescence T-jump can be assigned to the loop backbone dihedral angle flipping. Furthermore, we can predict that a Trp354 conformational conversion associated with the loop movements would occur on the tens of nanoseconds time scale, to be verified by future UVRR T-jump studies.

Background: Phytases are enzymes capable of degrading phytic acid and used in animal feed supplementation in order to improve digestibility through the release of minerals such as phosphorus., Objective: The main goal of this study was to express and characterize a Yersinia intermedia phytase expressed in Escherichia coli cells., Methods: The Y. intermedia phytase gene was synthesized and overexpressed in Escherichia coli cells. The phytase recombinante (rPHY) was purified to homogeneity using a Ni-NTA column. The biochemical and biophysical properties of the rPHY were measured in order to fully characterize the recombinant enzyme. The following patents database were consulted: Espacenet, USPTO, LATIPAT, Patent Scope, WIPO and Google Patents., Results: The results showed that the rPHY is active at 37-40ºC and presented an optimal pH and temperature of 8.0 and 40°C, respectively. The phytase rPHY was activated by Cu2+ ion and showed resistance to trypsin and pepsin, retaining 55% of the activity at the ratio of 0.02. Furthermore, the dissociation constant (Kd = 1.1150 ± 0.0087 mM), as estimated by a fluorescence binding assay, suggests a medium affinity of the enzyme with the substrate., Conclusion: The results of this article can be considered as innovative and for this reason, they were protected by Intellectual Property Law in Brazil. Take together, the biochemical properties of the rPHY could be useful in future for its industrial application of this enzyme as an additive in the monogastric feed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Phytases are phosphohydrolases that initiate the sequential hydrolysis of phosphate from phytate, which is the main storage form of phosphorous in numerous plant seeds, especially in cereals and grains. Phytate is indigestible for most monogastric animals, such as poultry, swine, fish, and humans; therefore, microbial phytases have been widely used in plant (specially soy)-based animal feeding to improve nutrition by enhanced phosphorus, mineral, and trace element absorption, and reducing phosphorus pollution by animal waste. Most phytases used as animal feed additives have an acid pH optimum (pH 2.5 and 5.5 for Aspergillus and pH 4.5 for E. coli phytases) and show a sharp decrease in performance at neutral pH, correlating with intestinal digestion. Directed evolution of phytases has been previously reported to improve enzyme thermostability, pH, or specific activity. In this manuscript, we report a directed evolution campaign of the highly active bacterial phytase from Yersinia mollaretii (YmPh) towards a broadened pH activity spectrum. Directed evolution identified the key positions T44 and K45 for increased YmPh activity at neutral pH. Both positions are located in the active site loop of the phytase and have a synergistic effect on activity with a broadened pH spectrum. Kinetic characterization of the improved variants, YmPh-M10 and -M16, showed up to sevenfold increased specific activity and up to 2.2-fold reduced K half at pH 6.6 under screening conditions compared to Yersinia mollaretii phytase wild type (YmPhWT).

To study factors that affect WPD-loop motion in protein tyrosine phosphatases (PTPs), a chimera of PTP1B and YopH was created by transposing the WPD loop from PTP1B to YopH. Several subsequent mutations proved to be necessary to obtain a soluble, active enzyme. That chimera, termed chimera 3, retains productive WPD-loop motions and general acid catalysis with a pH dependency similar to that of the native enzymes. Kinetic isotope effects show the mechanism and transition state for phosphoryl transfer are unaltered. Catalysis of the chimera is slower than that of either of its parent enzymes, although its rate is comparable to those of most native PTPs. X-ray crystallography and nuclear magnetic resonance were used to probe the structure and dynamics of chimera 3. The chimera's structure was found to sample an unproductive hyper-open conformation of its WPD loop, a geometry that has not been observed in either of the parents or in other native PTPs. The reduced catalytic rate is attributed to the protein's sampling of this conformation in solution, reducing the fraction in the catalytically productive loop-closed conformation.

Human infections by the intracellular bacterial pathogen Legionella pneumophila result in a severe form of pneumonia, the Legionnaire's disease. L. pneumophila utilizes a Type IVb secretion (T4bS) system termed "dot/icm" to secrete protein effectors to the host cytoplasm. The dot/icm system is powered at least in part by a functionally critical AAA+ ATPase, a protein called DotB, thought to belong to the VirB11 family of proteins. Here we present the crystal structure of DotB at 3.19 Å resolution, in its hexameric form. We observe that DotB is in fact a structural intermediate between VirB11 and PilT family proteins, with a PAS-like N-terminal domain coupled to a RecA-like C-terminal domain. It also shares critical structural elements only found in PilT. The structure also reveals two conformers, termed α and β, with an αβαβαβ configuration. The existence of α and β conformers in this class of proteins was confirmed by solving the structure of DotB from another bacterial pathogen, Yersinia, where, intriguingly, we observed an ααβααβ configuration. The two conformers co-exist regardless of the nucleotide-bound states of the proteins. Our investigation therefore reveals that these ATPases can adopt a wider range of conformational states than was known before, shedding new light on the extraordinary spectrum of conformations these ATPases can access to carry out their function. Overall, the structure of DotB provides a template for further rational drug design to develop more specific antibiotics to tackle Legionnaire's disease. PDB Code(s): Will; be; provided., (© 2018 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

Bovine pancreatic ribonuclease (RNase A) is the founding member of the RNase A superfamily. Members of this superfamily of ribonucleases have high sequence diversity, but possess a similar structural fold, together with a conserved His-Lys-His catalytic triad and structural disulfide bonds. Until recently, RNase A proteins had exclusively been identified in eukaryotes within vertebrae. Here, we discuss the discovery by Batot et al. of a bacterial RNase A superfamily member, CdiA-CT Ykris : a toxin that belongs to an inter-bacterial competition system from Yersinia kristensenii. CdiA-CT Ykris exhibits the same structural fold as conventional RNase A family members and displays in vitro and in vivo ribonuclease activity. However, CdiA-CT Ykris shares little to no sequence similarity with RNase A, and lacks the conserved disulfide bonds and catalytic triad of RNase A. Interestingly, the CdiA-CT Ykris active site more closely resembles the active site composition of various eukaryotic endonucleases. Despite lacking sequence similarity to eukaryotic RNase A family members, CdiA-CT Ykris does share high sequence similarity with numerous Gram-negative and Gram-positive bacterial proteins/domains. Nearly all of these bacterial homologs are toxins associated with virulence and bacterial competition, suggesting that the RNase A superfamily has a distinct bacterial subfamily branch, which likely arose by way of convergent evolution. Finally, RNase A interacts directly with the immunity protein of CdiA-CT Ykris , thus the cognate immunity protein for the bacterial RNase A member could be engineered as a new eukaryotic RNase A inhibitor.

Susceptibility to proteases usually limits the application of phytase. We sought to improve the pepsin and trypsin resistance of YeAPPA from Yersinia enterocolitica and YkAPPA from Y. kristensenii by optimizing amino acid polarity and charge. The predicted pepsin/trypsin cleavage sites F89/K226 in pepsin/trypsin-sensitive YeAPPA and the corresponding sites (F89/E226) in pepsin-sensitive but trypsin-resistant YkAPPA were substituted with S and H, respectively. Six variants were produced in Pichia pastoris for catalytic and biochemical characterization. F89S, E226H, and F89S/E226H elevated pepsin resistance and thermostability and K226H and F89S/K226H improved pepsin and trypsin resistance and stability at 60 °C and low pH. All the variants increased the ability of the proteins to hydrolyze phytate in corn meal by 2.6-14.9-fold in the presence of pepsin at 37 °C and low pH. This study developed a genetic manipulation strategy specific for pepsin/trypsin-sensitive phytases that can improve enzyme tolerance against proteases and heat and benefit the food and feed industry in a cost-effective way.

Contact-dependent growth inhibition (CDI) is an important mechanism of inter-bacterial competition found in many Gram-negative pathogens. CDI+ cells express cell-surface CdiA proteins that bind neighboring bacteria and deliver C-terminal toxin domains (CdiA-CT) to inhibit target-cell growth. CDI+ bacteria also produce CdiI immunity proteins, which specifically neutralize cognate CdiA-CT toxins to prevent self-inhibition. Here, we present the crystal structure of the CdiA-CT/CdiIYkris complex from Yersinia kristensenii ATCC 33638. CdiA-CTYkris adopts the same fold as angiogenin and other RNase A paralogs, but the toxin does not share sequence similarity with these nucleases and lacks the characteristic disulfide bonds of the superfamily. Consistent with the structural homology, CdiA-CTYkris has potent RNase activity in vitro and in vivo. Structure-guided mutagenesis reveals that His175, Arg186, Thr276 and Tyr278 contribute to CdiA-CTYkris activity, suggesting that these residues participate in substrate binding and/or catalysis. CdiIYkris binds directly over the putative active site and likely neutralizes toxicity by blocking access to RNA substrates. Significantly, CdiA-CTYkris is the first non-vertebrate protein found to possess the RNase A superfamily fold, and homologs of this toxin are associated with secretion systems in many Gram-negative and Gram-positive bacteria. These observations suggest that RNase A-like toxins are commonly deployed in inter-bacterial competition., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Strong resistance to proteolytic attack is important for feed enzymes. Here, we selected three predicted pepsin cleavage sites, L99, L162, and E230 (numbering from the initiator M of premature proteins), in pepsin-sensitive HAP phytases YkAPPA from Yersinia kristensenii and YeAPPA from Y. enterocolitica, which corresponded to L99, V162, and D230 in pepsin-resistant YrAPPA from Y. rohdei. We constructed mutants with different side chain structures at these sites using site-directed mutagenesis and produced all enzymes in Escherichia coli for catalytic and biochemical characterization. The substitutions E230G/A/P/R/S/T/D, L162G/A/V, L99A, L99A/L162G, and L99A/L162G/E230G improved the pepsin resistance. Moreover, E230G/A and L162G/V conferred enhanced pepsin resistance on YkAPPA and YeAPPA, increased their catalytic efficiency 1.3-2.4-fold, improved their stability at 60 °C and pH 1.0-2.0 and alleviated inhibition by metal ions. In addition, E230G increased the ability of YkAPPA and YeAPPA to hydrolyze phytate from corn meal at a high pepsin concentration and low pH, which indicated that optimization of the pepsin cleavage site side chains may enhance the pepsin resistance, improve the stability at acidic pH, and increase the catalytic activity. This study proposes an efficient approach to improve enzyme performance in monogastric animals fed feed with a high phytate content., Competing Interests: The authors declare no competing financial interests.

Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: ( i ) identification of conserved and highly variable regions; ( ii ) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; ( iii ) in vitro DNA recombination of synthetic genes; and ( iv ) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering-guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.

Yersinia sp. bacteria owe their viability and pathogenic virulence to the YopH factor, which is a highly active bacterial protein tyrosine phosphatase. Inhibition of YopH phosphatase results in the lack of Yersinia sp. pathogenicity. We have previously described that aurintricarboxylic acid inhibits the activity of YopH at nanomolar concentrations and represents a unique mechanism of YopH inactivation due to a redox process. This work is a continuation of our previous studies. Here we show that modifications of the structure of aurintricarboxylic acid reduce the ability to inactivate YopH and lead to higher cytotoxicity. In the present paper we examine the inhibitory properties of aurintricarboxylic acid analogues, such as eriochrome cyanine R (ECR) and pararosaniline. Computational docking studies we report here indicate that ATA analogues are not precluded to bind in the YopH active site and in all obtained binding conformations ECR and pararosaniline bind to YopH active site. The free binding energy calculations show that ECR has a stronger binding affinity to YopH than pararosaniline, which was confirmed by experimental YopH enzymatic activity studies. We found that ATA analogues can reversibly reduce the enzymatic activity of YopH, but possess weaker inhibitory properties than ATA. The ATA analogues induced inactivation of YopH is probably due to oxidative mechanism, as pretreatment with catalase prevents from inhibition. We also found that ATA analogues significantly decrease the viability of macrophage cells, especially pararosaniline, while ATA reveals only slight effect on cell viability., Competing Interests: The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Background: E.coli type II L-asparaginase is widely used for treatment of acute lymphoblastic leukemia. However, serious side effects such as allergic or hypersensitivity reactions are common for L-asparaginase treatment. Methods for minimizing immune response on L-asparaginase treatment in human include bioengeneering of less immunogenic version of the enzyme or utilizing the homologous enzymes of different origin. To rationalize these approaches we compared immunogenicity of L-asparaginases from five bacterial organisms and performed sequence-structure analysis of the presumable epitope regions., Methods: IgG and IgM immune response in C57B16 mice after immunization with Wollinella succinogenes type II (WsA), Yersinia pseudotuberculosis type II (YpA), Erwinia carotovora type II (EwA), and Rhodospirillum rubrum type I (RrA) and Escherichia coli type II (EcA) L-asparaginases was evaluated using standard ELISA method. The comparative bioinformatics analysis of structure and sequence of the bacterial L-asparaginases presumable epitope regions was performed., Results: We showed different immunogenic properties of five studied L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin as a second-line treatment. Studied L-asparaginases might be placed in the following order based on the immunogenicity level: YpA > RrA, WsA ≥ EwA > EcA. Most significant cross-immunogenicity was shown between EcA and YpA. We propose that a long N-terminus of YpA enzyme enriched with charged aminoacids and tryptophan could be a reason of higher immunogenicity of YpA in comparison with other considered enzymes. Although the recognized structural and sequence differences in putative epitope regions among five considered L-asparaginases does not fully explain experimental observation of the immunogenicity of the enzymes, the performed analysis set the foundation for further research in this direction., Conclusions: The performed studies showed different immunogenic properties of L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin. The preferable enzymes for the second line treatment are WsA, RrA, or EwA.

Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE,  an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The "best" combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes.

N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution.

Pathogenic Yersinia species evolved a type III secretion system that injects a set of effectors into the host cell cytosol to promote infection. One of these effectors, Yersinia protein kinase A (YpkA), is a multidomain effector that harbours a Ser/Thr kinase domain and a guanine dissociation inhibitor (GDI) domain. The intercellular targets of the kinase and GDI domains of YpkA were identified to be Gαq and the small GTPases RhoA and Rac1, respectively, which synergistically induce cytotoxic effects on infected cells. In this study, we demonstrate that vasodilator-stimulated phosphoprotein (VASP), which is critical for regulation of actin assembly, cell adhesion and motility, is a direct substrate of YpkA kinase activity. Ectopic co-expression of YpkA and VASP in HEK293T cells leads to the phosphorylation of VASP at S157, and YpkA kinase activity is essential for VASP phosphorylation at this site. Moreover, YpkA directly phosphorylates VASP in in vitro kinase assay. YpkA-mediated VASP phosphorylation significantly inhibits actin polymerization and promotes the disruption of actin cytoskeleton, which inhibits the phagocytosis. Taken together, our study found a novel molecular mechanism used by YpkA to disrupt cytoskeleton dynamics, thereby promoting the anti-phagocytosis ability of pathogenic Yersiniae., (© 2014 John Wiley & Sons Ltd.)

Nitroreductases (NRs) and ene-reductases (ERs) both utilize flavin mononucleotide cofactors but catalyze distinct reactions. NRs reduce nitroaromatics, whereas ERs reduce unsaturated C=C double bonds, and these functionalities are known to somewhat overlap. Recent studies on the ER xenobiotic reductase A (XenA) from Pseudomonas putida demonstrated the possibility of increasing NR activity with active site modifications. Structural comparison between NRs and ERs led us to hypothesize that active site cavity size plays an important role in determining enzyme functionality. Residues of ER KYE1 from Kluyveromyces lactis were selected to increase the binding pocket size, compensate for hydrogen bonding pattern changes, and eliminate ER activity. Single variants were screened, and promising mutations were combined. Variant F296A/Y275A showed a 100-fold improvement in NR specific activity over wild-type, and variant H191A/F296A/Y375A exhibited complete conversion to a NR., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Screening throughput is a key in directed evolution experiments and enzyme discovery. Here, we describe a high-throughput screening platform based on a coupled reaction of glucose oxidase and a hydrolase (Yersinia mollaretii phytase [YmPh]). The coupled reaction produces hydroxyl radicals through Fenton's reaction, acting as initiator of poly(ethyleneglycol)-acrylate-based polymerization incorporating a fluorescent monomer. As a consequence, a fluorescent hydrogel is formed around Escherichia coli cells expressing active YmPh. We achieve five times enrichment of active cell population through flow cytometry analysis and sorting of mixed populations. Finally, we validate the performance of the fluorescent polymer shell (fur-shell) technology by directed phytase evolution that yielded improved variants starting from a library containing 10(7) phytase variants. Thus, fur-shell technology represents a rapid and nonlaborious way of identifying the most active variants from vast populations, as well as a platform for generation of polymer-hybrid cells for biobased interactive materials., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

YopH is a protein tyrosine phosphatase that functions as a required virulence factor in Yersinia. Here we report the backbone resonance assignments for a point mutant of the C-terminal catalytic domain of YopH.

Directed evolution of Yersinia mollaretii phytase (Ymphytase) yielded an improved variant SM2P3E4 (also named M1; D52N, T77K, K139E, G187S, V298M) in our previous study. Variant M1 retained high specific activity (993U/mg; equivalent to 93% of wild-type activity) and improved thermal resistance (T50 improved by 1.5°C compared to wild-type at 58°C; 20min incubation time), making variant M1 an attractive enzyme for industrial applications. Recently, the OmniChange method was developed for multi-site saturation mutagenesis. The five sites identified in variant M1 were subjected to OmniChange saturation in order to explore whether a variant with higher activity, higher thermal resistance, and higher resistance at low pH (2-3h incubation was performed to mimic the gastric residence time of phytase) could be identified. Screening of a small library of 1100 clones, covering <0.004% of the theoretical sequence space of 3.35×10(7) variants, yielded a Ymphytase variant with 32% improved residual activity (58°C for 20min), 2°C increased apparent melting temperature (Tm), and 2-fold higher pH stability (pH 2.8; 3h incubation time) when compared to the wild-type Ymphytase. Compared to the M1 variant, the pH stability (pH 2.8; 3h incubation time) was improved by 3-fold, and thermal resistance as well as activity was improved slightly (residual activity: 32% compared to 20%; apparent Tm: 2°C compared to 1.5°C; activity difference <4%)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Microbes are incessantly challenged by both biotic and abiotic stressors threatening their existence. Therefore, bacterial pathogens must possess mechanisms to successfully subvert host immune defenses as well as overcome the stress associated with host-cell encounters. To achieve this, bacterial pathogens typically experience a genetic re-programming whereby anti-host/stress factors become expressed and eventually translated into effector proteins. In that vein, the bacterial host-cell induced stress-response is similar to any other abiotic stress to which bacteria respond by up-regulating specific stress-responsive genes. Following the stress encounter, bacteria must degrade unnecessary stress responsive transcripts through RNA decay mechanisms. The three pathogenic yersiniae (Yersinia pestis, Y. pseudo-tuberculosis, and Y. enterocolitica) are all psychrotropic bacteria capable of growth at 4°C; however, cold growth is dependent on the presence of an exoribonuclease, polynucleotide phosphorylase (PNPase). PNPase has also been implicated as a virulence factor in several notable pathogens including the salmonellae, Helicobacter pylori, and the yersiniae [where it typically influences the type three secretion system (TTSS)]. Further, PNPase has been shown to associate with ribonuclease E (endoribonuclease), RhlB (RNA helicase), and enolase (glycolytic enzyme) in several Gram-negative bacteria forming a large, multi-protein complex known as the RNA degradosome. This review will highlight studies demonstrating the influence of PNPase on the virulence potentials and stress responses of various bacterial pathogens as well as focusing on the degradosome-dependent and -independent roles played by PNPase in yersiniae stress responses.

The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia pestis and a potential antibacterial drug target. Here we report our studies of screening for small molecule inhibitors of YopH using both high throughput and in silico approaches. The identified inhibitors represent a diversity of chemotypes and novel pTyr mimetics, providing a starting point for further development and fragment-based design of multi-site binding inhibitors. We demonstrate that the applications of high throughput and virtual screening, when guided by structural binding mode analysis, is an effective approach for identifying potent and selective inhibitors of YopH and other protein phosphatases for rational drug design., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073 U/mg which is ∼10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ∼8,400 clones) a phytase variant with ∼20% improved thermostability (58°C for 20 min; residual activity wild type ∼34%; variant ∼53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993 U/mg).

Yersinia entomophaga MH96 is a native New Zealand soil bacterium that secretes a large ABC-type protein toxin complex, Yen-Tc, similar to those produced by nematode-associated bacteria such as Photorhabdus luminescens. Y. entomophaga displays an exceptionally virulent pathogenic phenotype in sensitive insect species, causing death within 72 h of infection. Because of this phenotype, there is intrinsic interest in the mechanism of action of Yen-Tc, and it also has the potential to function as a novel class of biopesticide. We have identified genes that encode chitinases as part of the toxin complex loci in Y. entomophaga MH96, P. luminescens, Photorhabdus asymbiotica and Xenorhabdus nematophila. Furthermore, we have shown that the secreted toxin complex from Y. entomophaga MH96 includes two chitinases as an integral part of the complex, a feature not described previously in other ABC toxins and possibly related to the severe disease caused by this bacterium. We present here the structure of the Y. entomophaga MH96 Chi1 chitinase, determined by X-ray crystallography to 1.74 Å resolution, and show that a ring of five symmetrically arranged lobes on the surface of the Yen-Tc toxin complex structure, as determined by single-particle electron microscopy, provides a good fit to the Chi1 monomer. We also confirm that the isolated chitinases display endochitinase activity, as does the complete toxin complex., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Accumulation of gyrase cleavage complex in Escherichia coli from the action of quinolone antibiotics induces an oxidative damage cell death pathway. The oxidative cell death pathway has also been shown to be involved in the lethality following accumulation of cleavage complex formed by bacterial topoisomerase I with mutations that result in defective DNA religation., Methods: A high copy number plasmid clone spanning the upp-purMN region was isolated from screening of an E. coli genomic library and analyzed for conferring increased survival rates following accumulation of mutant topoisomerase I proteins as well as treatment with the gyrase inhibitor norfloxacin., Results: Analysis of the intergenic region upstream of purM demonstrated a novel mechanism of resistance to the covalent protein-DNA cleavage complex through titration of the cellular transcription regulators FNR and PurR responsible for oxygen sensing and repression of purine nucleotide synthesis respectively. Addition of adenine to defined growth medium had similar protective effect for survival following accumulation of topoisomerase cleavage complex, suggesting that increase in purine level can protect against cell death., Conclusions: Perturbation of the global regulator FNR and PurR functions as well as increase in purine nucleotide availability could affect the oxidative damage cell death pathway initiated by topoisomerase cleavage complex.

Asymmetric trans-bioreduction of activated alkenes by KYE1 from Kluyveromyces lactis and Yers-ER from Yersinia bercovieri, two ene-reductases from the Old Yellow Enzyme family, showed a broad substrate spectrum with a moderate to excellent degree of stereoselectivity. Both substrate- and enzyme-based stereocontrols were observed to furnish opposite stereoisomeric products. The effects of organic solvents on enzyme activity and stereoselectivity were outlined in this study, where two-phase systems hexane and toluene are shown to sustain bioreduction efficiency even at high organic solvent content.

We have previously demonstrated that Yersinia ruckeri resists cecropin B in an inducible manner. In this study, we sought to identify the molecular changes responsible for the inducible cecropin B resistance of Y. ruckeri. Differences in gene expression associated with the inducible resistance were investigated. Cultures of Y. ruckeri were exposed to a sublethal concentration of cecropin B and resultant changes in the messenger RNA population of the bacteria were assayed using the differential display reverse transcription polymerase chain reaction (DD-RT-PCR). A single band was consistently increased in intensity in all repeats of the experiment. The band was excised, cloned, sequenced, and used to screen a Y. ruckeri genomic DNA library. The DD-RT-PCR fragment shared 100% identity to the cDNA sequence of an ATP-dependent endonuclease of the overcome lysogenization defect (OLD) family of Y. ruckeri 29473. The genomic clone that was recovered was not identical to the DD-RT-PCR clone, but harbored a gene for a secreted endonuclease 1 (nucM) homologue. It was determined that transcription of the gene was upregulated following exposure to cecropin B via RT-PCR. Furthermore, an increase in the nuclease activity of culture supernatants of Y. ruckeri following exposure to cecropin B was demonstrated. These findings demonstrate that cecropin B exposure increases the expression of at least two endonucleases in Y. ruckeri. The production and secretion of an endonuclease by Y. ruckeri in response to an antimicrobial peptide indicates the involvement of both intracellular and extracellular DNA in the toxic effects of cecropin B.

We report on the design and evaluation of novel cyclic peptides targeting the N-terminal domain of the protein tyrosine phosphatase YopH from Yersinia. Cyclic peptides have been designed based on a short sequence from the protein SKAP-HOM [DE(pY)DDPF (pY=phosphotyrosine)], and they all contain the motif DEZXDPfK (where Z is a phosphotyrosine or a non-hydrolyzable phosphotyrosine mimetic, X is an aspartic acid or a leucine and f is a d-phenylalanine). These peptides present a 'head to tail' architecture, enabling cyclization through formation of an amide bond in between the side chains of the first aspartic acid and the lysine residues. Chemical shift perturbation studies have been carried out to monitor the binding of these peptides to the N-terminal domain of YopH. Peptides containing a phosphotyrosine moiety exhibit binding affinities in the low micromolar range; substitution of the phosphotyrosine with one of its non-hydrolyzable derivatives dramatically reduces the binding affinities. These preliminary studies may pave the way for the discovery of more potent and selective peptide-based ligands of the YopH N-terminal domain which could be further investigated for their ability to inhibit Yersiniae infections., (© 2010 John Wiley & Sons A/S.)

Plague, one of the most devastating diseases in human history, is caused by the bacterium Yersinia pestis. The bacteria use a syringe-like macromolecular assembly to secrete various toxins directly into the host cells they infect. One such Yersinia outer protein, YopJ, performs the task of dampening innate immune responses in the host by simultaneously inhibiting the MAPK and NFkappaB signaling pathways. YopJ catalyzes the transfer of acetyl groups to serine, threonine, and lysine residues on target proteins. Acetylation of serine and threonine residues prevents them from being phosphorylated thereby preventing the activation of signaling molecules on which they are located. In this study, we describe the requirement of a host-cell factor for full activation of the acetyltransferase activity of YopJ and identify this activating factor to be inositol hexakisphosphate (IP(6)). We extend the applicability of our results to show that IP(6) also stimulates the acetyltransferase activity of AvrA, the YopJ homologue from Salmonella typhimurium. Furthermore, an IP(6)-induced conformational change in AvrA suggests that IP(6) acts as an allosteric activator of enzyme activity. Our results suggest that YopJ-family enzymes are quiescent in the bacterium where they are synthesized, because bacteria lack IP(6); once injected into mammalian cells by the pathogen these toxins bind host cell IP(6), are activated, and deregulate the MAPK and NFkappaB signaling pathways thereby subverting innate immunity.

Microbial pathogens exploit the ubiquitin system to facilitate infection and manipulate the immune responses of the host. In this study, susceptibility to Yersinia enterocolitica and Yersinia pseudotuberculosis invasion was found to be increased upon overexpression of the deubiquitinating enzyme otubain 1 (OTUB1), a member of the ovarian tumour domain-containing protein family. Conversely, OTUB1 knockdown interfered with Yersinia invasion in HEK293T cells as well as in primary monocytes. This effect was attributed to a modulation of bacterial uptake. We demonstrate that the Yersinia-encoded virulence factor YpkA (YopO) kinase interacts with a post-translationally modified form of OTUB1 that contains multiple phosphorylation sites. OTUB1, YpkA and the small GTPase ras homologue gene family member A (RhoA) were found to be part of the same protein complex, suggesting that RhoA levels are modulated by OTUB1. Our results show that OTUB1 is able to stabilize active RhoA prior to invasion, which is concomitant with an increase in bacterial uptake. This effect is modulated by post-translational modifications of OTUB1, suggesting a new entry point for manipulating Yersinia interactions with the host.

A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The specific activity was 137 micromol/min/mg. The Michaelis constant (k(m) value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 degrees C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.

A new phytase (APPA) with optimum pH 2.5--substantially lower than that of most of microbial phytases (pH 4.5-6.0)--was cloned from Yersinia frederiksenii and heterologously expressed in Escherichia coli. Containing the highly conserved motifs typical of histidine acid phosphatases, APPA has the highest identity (84%) to the Yersinia intermedia phytase (optimal pH 4.5), a member of histidine acid phosphatase family. Based on sequence alignment and molecular modeling of APPA and related phytases, APPA has only one divergent residue, Ser51, in close proximity to the catalytic site. To understand the acidic adaptation of APPA, five mutants (S51A, S51T, S51D, S51K, and S51I) were constructed by site-directed mutagenesis, expressed in E. coli, purified, and characterized. Mutants S51T and S51I exhibited a shift in the optimal pH from 2.5 to 4.5 and 5.0, respectively, confirming the role of Ser51 in defining the optimal pH. Thus, a previously unrecognized factor other than electrostatics--presumably the side-chain structure near the active site--contributes to the optimal pH for APPA activity. Compared with wild-type APPA, mutant S51T showed higher specific activity, greater activity over pH 2.0-5.5, and increased thermal and acid stability. These properties make S51T a better candidate than the wild-type APPA for use in animal feed.

Nucleoside 5'-diphosphate-X hydrolases are interesting enzymes to study due to their varied activities and structure-function relationships and the roles they play in the disposal, assimilation, and modulation of the effects of their substrates. Few of these enzymes with a preference for CDP-alcohols are known. In Yersinia intermedia suspensions prepared from cultures on Columbia agar with 5% sheep blood, we found a CDP-alcohol hydrolase liberated to Triton X-100-containing medium. Growth at 25 degrees C was deemed optimum in terms of the enzyme-activity yield. The purified enzyme also displayed 5'-nucleotidase, UDP-sugar hydrolase, and dinucleoside-polyphosphate hydrolase activities. It was identified as the protein product (UshA(Yi)) of the Y. intermedia ushA gene (ushA(Yi)) by its peptide mass fingerprint and by PCR cloning and expression to yield active enzyme. All those activities, except CDP-alcohol hydrolase, have been shown to be the properties of UshA of Escherichia coli (UshA(Ec)). Therefore, UshA(Ec) was expressed from an appropriate plasmid and tested for CDP-alcohol hydrolase activity. UshA(Ec) and UshA(Yi) behaved similarly. Besides being the first study of a UshA enzyme in the genus Yersinia, this work adds CDP-alcohol hydrolase to the spectrum of UshA activities and offers a novel perspective on these proteins, which are viewed here for the first time as highly efficient enzymes with k(cat)/K(m) ratios near the theoretical maximum level of catalytic activities. The results are discussed in the light of the known structures of UshA(Ec) conformers and the respective homology models constructed for UshA(Yi), and also in relation to possible biological functions. Interestingly, every Yersinia species with a sequenced genome contains an intact ushA gene, except Y. pestis, which in all its sequenced biovars contains a ushA gene inactivated by frameshift mutations.

Two novel phytase genes belonging to the histidine acid phosphatase family were cloned from Yersinia rohdei and Y. pestis and expressed in Pichia pastoris. Both the recombinant phytases had high activity at pH 1.5-6.0 (optimum pH 4.5) with an optimum temperature of 55 degrees C. Compared with the major commercial phytases from Aspergillus niger, Escherichia coli, and a potential commercial phytase from Y. intermedia, the Y. rohdei phytase was more resistant to pepsin, retained more activity under gastric conditions, and released more inorganic phosphorus (two to ten times) from soybean meal under simulated gastric conditions. These superior properties suggest that the Y. rohdei phytase is an attractive additive to animal feed. Our study indicated that, in order to better hydrolyze the phytate and release more inorganic phosphorus in the gastric passage, phytase should have high activity and stability, simultaneously, at low pH and high protease concentration.

Yersinia spp. use a type 3 secretion system (T3SS) to directly inject six proteins into macrophages, and any impairment of this process results in a profound reduction in virulence. We previously showed that the exoribonuclease polynucleotide phosphorylase (PNPase) was required for optimal T3SS functioning in Yersinia pseudotuberculosis and Yersinia pestis. Here we report that Y. pseudotuberculosis cells with reduced RNase E activity are likewise impaired in T3SS functioning and that phenotypically they resemble Delta pnp cells. RNase E does not affect expression levels of the T3SS substrates but instead, like PNPase, regulates a terminal event in the secretion pathway. This similarity, together with the fact that RNase E and PNPase can be readily copurified from Y. pseudotuberculosis cell extracts, suggests that these two RNases regulate T3SS activity through a common mechanism. This is the first report that RNase E activity impacts the T3SS as well as playing a more general role in infectivity.

Yersinia spp. pathogens use their type III secretion system to translocate effectors that manipulate host signaling pathways during infection. Although molecular targets for five of the six known Yersinia effectors are known, the target for the serine/threonine kinase domain of Yersinia protein kinase A (YpkA) has remained elusive. Recently, Navarro et al. (2007) demonstrated that YpkA phosphorylates Galphaq, and inhibits Galphaq-mediated signaling. Inhibition by YpkA could contribute to one of the most documented symptoms of Yersinia pestis infection, extensive bleeding.

Since convergent evolution of enzyme active sites was first identified in serine proteases, other individual instances of this phenomenon have been documented. However, a systematic analysis assessing the frequency of this phenomenon across enzyme space is still lacking. This work uses the Query3d structural comparison algorithm to integrate for the first time detailed knowledge about catalytic residues, available through the Catalytic Site Atlas (CSA), with the evolutionary information provided by the Structural Classification of Proteins (SCOP) database. This study considers two modes of convergent evolution: (i) mechanistic analogues which are enzymes that use the same mechanism to perform related, but possibly different, reactions (considered here as sharing the first three digits of the EC number); and (ii) transformational analogues which catalyse exactly the same reaction (identical EC numbers), but may use different mechanisms. Mechanistic analogues were identified in 15% (26 out of 169) of the three-digit EC groups considered, showing that this phenomenon is not rare. Furthermore 11 of these groups also contain transformational analogues. The catalytic triad is the most widespread active site; the results of the structural comparison show that this mechanism, or variations thereof, is present in 23 superfamilies. Transformational analogues were identified for 45 of the 951 four-digit EC numbers present within the CSA and about half of these were also mechanistic analogues exhibiting convergence of their active sites. This analysis has also been extended to the whole Protein Data Bank to provide a complete and manually curated list of the all the transformational analogues whose structure is classified in SCOP. The results of this work show that the phenomenon of convergent evolution is not rare, especially when considering large enzymatic families.

Pathogenic species of the genus Yersinia suppress and reorient the immune system to infect lymphatic tissues, inner organs and at times also the vasculature. For this purpose yersiniae employ a type III secretion system to translocate effector proteins (Yersinia outer proteins; Yops) into immune cells. Yops often exert unique biochemical activities for modulating the activity of Rho GTP-binding proteins, focal adhesion proteins, inflammatory pathways and cell survival/apoptosis. In this review we will put emphasis on the biochemistry, cell- and infection biology of Yersinia effector Yops.

Yersinia spp. is currently an antibiotic resistance concern and a re-emerging disease. The essential virulence factor Yersinia protein kinase A (YpkA) contains a Ser/Thr kinase domain whose activity modulates pathogenicity. Here, we present an approach integrating a machine learning method, homology modeling, and multiple conformational high-throughput docking for the discovery of YpkA inhibitors. These first reported inhibitors of YpkA may facilitate studies of the pathogenic mechanism of YpkA and serve as a starting point for development of anti-plague drugs.

Previously, it was shown that optimal functioning of the Yersinia type III secretion system (T3SS) in cell culture infection assays requires the exoribonuclease polynucleotide phosphorylase (PNPase) and that normal T3SS activity could be restored in the Deltapnp strains by expressing just the approximately 70-aa S1 RNA-binding domain of PNPase. Here, it is shown that the Yersinia Deltapnp strain is less virulent in the mouse compared with the isogenic wild-type strain. To begin to understand what could be limiting T3SS activity in the absence of PNPase, T3SS-encoding transcripts and proteins in the YersiniaDeltapnp strains were analyzed. Surprisingly, it was found that the Deltapnp Yersinia strains possessed enhanced levels of T3SS-encoding transcripts and proteins compared with the wild-type strains. We then found that an S1 variant containing a disruption in its RNA-binding subdomain was inactive in terms of restoring normal T3SS activity. However, T3SS expression levels did not differ between Deltapnp strains expressing active and inactive S1 proteins, further showing that T3SS activity and expression levels, at least as related to PNPase and its S1 domain, are not linked. The results suggest that PNPase affects the expression and activity of the T3SS by distinct mechanisms and that the S1-dependent effect on T3SS activity involves an RNA intermediate.

Mitochondrial NADH:ubiquinone oxidoreductase is the largest and most complicated proton pump of the respiratory chain. Here we report the preparation and characterization of a subcomplex of complex I selectively lacking the flavoprotein part of the N-module. Removing the 51-kDa and the 24-kDa subunit resulted in loss of catalytic activity. The redox centers of the subcomplex could be reduced neither by NADH nor NADPH demonstrating that physiological electron input into complex I occurred exclusively via the N-module and that the NADPH binding site in the 39-kDa subunit and further potential nucleotide binding sites are isolated from the electron transfer pathway within the enzyme. Taking advantage of the selective removal of two of the eight iron-sulfur clusters of complex I and providing additional evidence by redox titration and site-directed mutagenesis, we could for the first time unambiguously assign cluster N1 of fungal complex I to mammalian cluster N1b.

The Yersinia protein tyrosine phosphatase (YopH) contains a loop of ten amino acids (the WPD loop) that covers the entrance of the active site of the enzyme during substrate binding. In this work the substrate mimicking competitive inhibitor p-nitrocatechol sulfate (PNC) is used as a probe of the active site. The dynamics of the WPD loop was determined by subjecting an equilibrated system containing YopH, PNC, and YopH bound to PNC to a laser induced temperature jump, and subsequently following the change in equilibrium due to the perturbation. Using this methodology the dynamics associated with substrate binding in YopH have been determined. These results indicate that substrate binding is coupled to the WPD loop motion, and WPD loop dynamics occur in the sub-millisecond time scale. The significance of these dynamic results is interpreted in terms of the catalytic cycle of the enzyme.

Background: The presence of beta-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the beta-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of beta-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India., Results: The enzymes, Bla-A (a constitutive class A penicillinase) and Bla-B (an inducible class C cephalosporinase) were found to be present in all the clinical and non-clinical strains of Y. intermedia and Y. frederiksenii by double disc diffusion method. The results showed differential expression of Bla-A as indicated by presence/absence of synergy whereas expression of Bla-B was quite consistent. The presence of these enzymes was also reflected in the high minimum inhibitory concentrations, MIC50 (126-1024 mg/L) and MIC90 (256-1024 mg/L) of beta-lactam antibiotics against these species. Restriction fragment length polymorphism (RFLP) revealed heterogeneity in both blaA and blaB genes of Y. intermedia and Y. frederiksenii. The blaA gene of Y. intermedia shared significant sequence identity (87-96%) with blaA of Y. enterocolitica biovars 1A, 1B and 4. The sequence identity of blaA of Y. frederiksenii with these biovars was 77-79%. The sequence identity of blaB gene of Y. intermedia and Y. frederiksenii was more (85%) with that of Y. enterocolitica biovars 1A, 1B and 2 compared to other species viz., Y. bercovieri, Y. aldovae and Y. ruckeri. Isoelectric focusing data further revealed that both Y. intermedia and Y. frederiksenii produced Bla-A (pI 8.7) and "Bla-B like" (pI 5.5-7.1) enzymes., Conclusion: Both Y. intermedia and Y. frederiksenii showed presence of blaA and blaB genes and unequivocal expression of the two beta-lactamases. Limited heterogeneity was detected in blaA and blaB genes as judged by PCR-RFLP. Phylogenetic relationships showed that the two species shared a high degree of identity in their bla genes. This is the first study reporting characteristics of beta-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii isolated from Asian region.

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject effector proteins (Yops) into host cells. The Yops down-regulate host cell functions through unique biochemical activities. YopO, a serine/threonine kinase required for Yersinia virulence, is activated by host cell actin via an unknown process. Here we show that YopO kinase is activated by formation of a 1:1 complex with monomeric (G) actin but is unresponsive to filamentous (F) actin. Two separate G-actin binding sites, one in the N-terminal kinase region (amino acids 89-440) and one in the C-terminal guanine nucleotide dissociation inhibitor-like region (amino acids 441-729) of YopO, were identified. Actin binding to both of these sites was necessary for effective autophosphorylation of YopO on amino acids Ser-90 and Ser-95. A S90A/S95A YopO mutant was strongly reduced in substrate phosphorylation, suggesting that autophosphorylation activates YopO kinase activity. In cells the kinase activity of YopO regulated rounding/arborization and was specifically required for inhibition of Yersinia YadA-dependent phagocytosis. Thus, YopO kinase is activated by a novel G-actin binding process, and this appears to be crucial for its anti-host cell functions.

Protein tyrosine phosphatases (PTPs) are important regulators of signal transduction pathways. Potent and selective PTP inhibitors are useful for probing these pathways and also may serve as drugs for the treatment of a variety of diseases including type 2 diabetes and infection by the bacterium Yersinia pestis. In this report Cu(I)-catalyzed 'click' cycloaddition reactions between azides and alkynes were employed to generate two sequential libraries of PTP inhibitors. In the first round library methyl 4-azidobenzoylformate was reacted with 56 mono- and diynes. After hydrolysis of the methyl esters, the resulting alpha-ketocarboxylic acids were assayed in crude form against the Yersinia PTP and PTP1B. Four compounds were selected for further evaluation, and one compound was chosen as the lead for generation of the second round library. This lead compound was modified by conversion of an alcohol into an azide group, and the resulting azide was reacted with the same 56 mono- and diynes that were used in the first generation library. After screening the crude inhibitors against the Yersinia PTP and PTP1B, four compounds were selected and evaluated in pure form against the Yersinia PTP, PTP1B, TCPTP, LAR, and CD45. The best bis(alpha-ketocarboxylic acid) inhibitor 34 had an IC(50) value of 550nM against the Yersinia PTP and an IC(50) value of 710nM against TCPTP. The most potent inhibitor containing a single alpha-ketocarboxylic acid group 32 had IC(50) values of 2.1, 5.7, and 2.6 microM against the Yersinia PTP, PTP1B, and TCPTP, respectively.