Purification, Characterization, and Structural Studies of a Sulfatase from Pedobacter yulinensis.

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications. We expressed and characterized a putative sulfatase (PyuS) from the bacterium Pedobacter yulinensis. PyuS contains the (C/S)XPXR sulfatase motif, where the Cys or Ser is post-translationally converted into a formylglycine residue (FGly). His-tagged PyuS was co-expressed in Escherichia coli with a formylglycine-generating enzyme (FGE) from Mycobacterium tuberculosis and purified. We obtained several crystal structures of PyuS, and the FGly modification was detected at the active site. The enzyme has sulfatase activity on aromatic sulfated substrates as well as phosphatase activity on some aromatic phosphates; however, PyuS did not have detectable activity on 17α-estradiol sulfate, cortisol 21-sulfate, or boldenone sulfate.

Production and Use of Recombinant Profilins Amb a 8, Art v 4, Bet v 2, and Phl p 12 for Allergenic Sensitization Studies.

Four recombinant (r) allergens (rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101) were successfully produced and used for sensitization studies. The allergens belong to the profilin family which is one of the most numerous allergen families. These four proteins represent allergens originating from pollen of weeds (rAmb a 8.0101 and rArt v 4.0101), tree (rBet v 2.0101) and grass (rPhl p 12.0101). The recombinant allergens were characterized using various biochemical and biophysical methods and tested for their ability to bind patient-derived antibodies. One hundred patients aged 2 to 50 years sensitized to pollen and plant-derived food allergens (IgE > 0.35 kU/L) were included. Sensitization to individual allergen sources and components of birch and timothy pollens was evaluated using multiparameter immunoblots. The presence of IgE to pollen-derived recombinant profilins rAmb a 8.0101, rArt v 4.0101, rBet v 2.0101, and rPhl p 12.0101 in serum was evaluated using ELISA method. The presence of IgE against pollen profilins was detected in 20 out of 100 studied patients. High correlation was seen between IgE ELISA results with individual pollen profilins. In summary, it was shown that the recombinant versions of the four allergenic profilins can be used for sensitization studies and for component-resolved allergy diagnostics.

Comparative studies of Aspergillus fumigatus 2-methylcitrate synthase and human citrate synthase.

Aspergillus fumigatus is a ubiquitous fungus that is not only a problem in agriculture, but also in healthcare. Aspergillus fumigatus drug resistance is becoming more prominent which is mainly attributed to the widespread use of fungicides in agriculture. The fungi-specific 2-methylcitrate cycle is responsible for detoxifying propionyl-CoA, a toxic metabolite produced as the fungus breaks down proteins and amino acids. The enzyme responsible for this detoxification is 2-methylcitrate synthase (mcsA) and is a potential candidate for the design of new anti-fungals. However, mcsA is very similar in structure to human citrate synthase (hCS) and catalyzes the same reaction. Therefore, both enzymes were studied in parallel to provide foundations for design of mcsA-specific inhibitors. The first crystal structures of citrate synthase from humans and 2-methylcitrate synthase from A. fumigatus are reported. The determined structures capture various conformational states of the enzymes and several inhibitors were identified and characterized. Despite a significant homology, mcsA and hCS display pronounced differences in substrate specificity and cooperativity. Considering that the active sites of the enzymes are almost identical, the differences in reactions catalyzed by enzymes are caused by residues that are in the vicinity of the active site and influence conformational changes of the enzymes.

Structural, Functional, and Immunological Characterization of Profilin Panallergens Amb a 8, Art v 4, and Bet v 2.

Ragweed allergens affect several million people in the United States and Canada. To date, only two ragweed allergens, Amb t 5 and Amb a 11, have their structures determined and deposited to the Protein Data Bank. Here, we present structures of methylated ragweed allergen Amb a 8, Amb a 8 in the presence of poly(l-proline), and Art v 4 (mugwort allergen). Amb a 8 and Art v 4 are panallergens belonging to the profilin family of proteins. They share significant sequence and structural similarities, which results in cross-recognition by IgE antibodies. Molecular and immunological properties of Amb a 8 and Art v 4 are compared with those of Bet v 2 (birch pollen allergen) as well as with other allergenic profilins. We purified recombinant allergens that are recognized by patient IgE and are highly cross-reactive. It was determined that the analyzed allergens are relatively unstable. Structures of Amb a 8 in complex with poly(l-proline)10 or poly(l-proline)14 are the first structures of the plant profilin in complex with proline-rich peptides. Amb a 8 binds the poly(l-proline) in a mode similar to that observed in human, mouse, and P. falciparum profilin·peptide complexes. However, only some of the residues that form the peptide binding site are conserved.

Structural and functional characterization of ß-cyanoalanine synthase from Tetranychus urticae.

Tetranychus urticae is a polyphagous spider mite that can feed on more than 1100 plant species including cyanogenic plants. The herbivore genome contains a horizontally acquired gene tetur10g01570 (TuCAS) that was previously shown to participate in cyanide detoxification. To understand the structure and determine the function of TuCAS in T. urticae, crystal structures of the protein with lysine conjugated pyridoxal phosphate (PLP) were determined. These structures reveal extensive TuCAS homology with the ß-substituted alanine synthase family, and they show that this enzyme utilizes a similar chemical mechanism involving a stable α-aminoacrylate intermediate in ß-cyanoalanine and cysteine synthesis. We demonstrate that TuCAS is more efficient in the synthesis of ß-cyanoalanine, which is a product of the detoxification reaction between cysteine and cyanide, than in the biosynthesis of cysteine. Also, the enzyme carries additional enzymatic activities that were not previously described. We show that TuCAS can detoxify cyanide using O-acetyl-L-serine as a substrate, leading to the direct formation of ß-cyanoalanine. Moreover, it catalyzes the reaction between the TuCAS-bound α-aminoacrylate intermediate and aromatic compounds with a thiol group. In addition, we have tested several compounds as TuCAS inhibitors. Overall, this study identifies additional functions for TuCAS and provides new molecular insight into the xenobiotic metabolism of T. urticae.

Variants of glycosyl hydrolase family 2 ß-glucuronidases have increased activity on recalcitrant substrates.

Glucuronidated drug metabolites can be quantified from urine samples by first hydrolyzing conjugates with ß-glucuronidase (ß-GUS) and then separating free drug molecules by liquid chromatography and mass spectrometry detection (LC-MS). To improve the activity and specificity of various ß-GUS, we designed enzyme chimeras and generated site-saturation variants based on structural analyses, then screened them for improved activity on drug metabolites important to clinical and forensic drug-testing laboratories. Often, an increase of activity on one substrate of interest was countered by loss of activity against another, and there was no strong correlation of activity on standard ß-glucuronidase substrates to activity on recalcitrant drug glucuronides. However, we discovered a chimera of two enzymes from different species of Aspergillus that displays a 27 % increase in activity on morphine-3-glucuronide than the parent proteins. Furthermore, mutations in the M-loop, which is a loop near the active site, resulted in numerous variants with dramatically increased rates of hydrolysis on drug glucuronides. Specifically, the M-loop variant Q451D/A452E of a ß-GUS from Brachyspira pilosicoli has a 50-fold and 25-fold increase in activity on the recalcitrant substrates codeine-6-glucuronide and dihydrocodeine-6-glucuronide, respectively, compared to the parent enzyme.

Sensitization to major Dermatophagoides pteronyssinus allergens in house dust mite allergic patients from North Eastern Poland developing rhinitis or asthma.

PURPOSE: Recognition of individual allergens by IgE is crucial for triggering symptoms in allergic rhinitis (AR) or asthmatic (AA) patients. House dust mite (HDM) allergy is frequent around the world, the sensitization profile to individual HDM allergens varies in individual HDM-allergic patients (APs). The aim of this study was to evaluate the pattern of IgE sensitization to three major Dermatophagoides pteronyssinus (Dp) allergens among patients from North Eastern Poland suffering from HDM-AR and/or AA. PATIENTS AND METHODS: The study was performed on 323 HDM-AR and/or AA patients and 106 controls (CG) including 30 healthy non-atopic subjects, 32 AR patients not sensitized to Dp and 44 non-atopic asthmatics. IgE levels to natural (n)Der p 1, nDer p 2, recombinant (r)Der p 2.0101 and rDer p 23 allergens were measured by ELISA. RESULTS: The majority of HDM-APs were sensitized to nDer p 1 (72.1%), nDer p 2 (81.7%), rDer p 2.0101 (78.3%) and rDer p 23 (70.9%). The frequency of positive results to individual allergens depended on clinical manifestations and the level of IgE to the whole Dp extract. In HDM-AA patients, reactivity to nDer p 1 and rDer p 23 was detected more frequently than in HDM-AR patients. The whole Dp extract completely inhibited IgE binding to nDer p 1 and nDer p 2 but only partially to rDer p 23. CONCLUSIONS: HDM-APs from North-Eastern Poland display sensitization profile to major allergens which is similarly observed in western Europe. HDM-based diagnostic and therapeutic products should include all major allergens.

Comparative structural and thermal stability studies of Cuc m 2.0101, Art v 4.0101 and other allergenic profilins.

Worldwide, more than one-third of the population suffers from allergies. A significant fraction of officially registered allergens originate from the profilin family of proteins. Profilins are small ubiquitous proteins which are found in plants, viruses and various eukaryotes including mammals. Although they are primarily regarded as minor allergens, profilins are important players in immunoglobulin E (IgE) cross-reactivity. However, in some populations profilins are recognized by IgE from at least 50% of patients allergic to a given allergen source. Cuc m 2.0101 is recognized by IgE in more than 80% of muskmelon-allergic patients. The recombinant isoallergen Cuc m 2.0101 was produced in significant quantities and its X-ray crystal structure was determined. In addition, a new Art v 4.0101 (mugwort profilin) structure was determined. The profilins Cuc m 2.0101 and Art v 4.0101 were compared in terms of their structure and thermal stability. Furthermore, structural similarities and IgE cross-reactivity between profilins from different sources are discussed to explain the molecular basis of various clinical syndromes involving this group of allergens. Special emphasis is placed on discussion of profilins' quaternary structures and their relation to biological function, as well as to protein allergenicity. Moreover, a potential impact of protein purification protocols on the structure of profilins is highlighted.

Structural and functional characterization of an intradiol ring-cleavage dioxygenase from the polyphagous spider mite herbivore Tetranychus urticae Koch.

Genome analyses of the polyphagous spider mite herbivore Tetranychus urticae (two-spotted spider mite) revealed the presence of a set of 17 genes that code for secreted proteins belonging to the "intradiol dioxygenase-like" subgroup. Phylogenetic analyses indicate that this novel enzyme family has been acquired by horizontal gene transfer. In order to better understand the role of these proteins in T. urticae, we have structurally and functionally characterized one paralog (tetur07g02040). It was demonstrated that this protein is indeed an intradiol ring-cleavage dioxygenase, as the enzyme is able to cleave catechol between two hydroxyl-groups using atmospheric dioxygen. The enzyme was characterized functionally and structurally. The active site of the T. urticae enzyme contains an Fe3+ cofactor that is coordinated by two histidine and two tyrosine residues, an arrangement that is similar to those observed in bacterial homologs. However, the active site is significantly more solvent exposed than in bacterial proteins. Moreover, the mite enzyme is monomeric, while almost all structurally characterized bacterial homologs form oligomeric assemblies. Tetur07g02040 is not only the first spider mite dioxygenase that has been characterized at the molecular level, but is also the first structurally characterized intradiol ring-cleavage dioxygenase originating from a eukaryote.

Impact of an N-terminal Polyhistidine Tag on Protein Thermal Stability.

For years, the use of polyhistidine tags (His-tags) has been a staple in the isolation of recombinant proteins in immobilized metal affinity chromatography experiments. Their usage has been widely beneficial in increasing protein purity from crude cell lysates. For some recombinant proteins, a consequence of His-tag addition is that it can affect protein function and stability. Functional proteins are essential in the elucidation of their biological, kinetic, structural, and thermodynamic properties. In this study, we determine the effect of N-terminal His-tags on the thermal stability of select proteins using differential scanning fluorimetry and identify that the removal of the His-tag can have both beneficial and deleterious effects on their stability.

Structural Characterization of a Eukaryotic Cyanase from Tetranychus urticae.

The two-spotted spider mite Tetranychus urticae is a polyphagous agricultural pest and poses a high risk to global crop production as it is rapidly developing pesticide resistance. Genomic and transcriptomic analysis has revealed the presence of a remarkable cyanase gene in T. urticae and related mite species within the Acariformes lineage. Cyanase catalyzes the detoxification of cyanate and is potentially an attractive protein target for the development of new acaricides. Phylogenetic analysis indicates that within the Acariformes, the cyanase gene originates from a single horizontal gene transfer event, which precedes subsequent speciation. Our structural studies presented here compare and contrast prokaryotic cyanases to T. urticae cyanase, which all form homodecamers and have conserved active site residues, but display different surface areas between homodimers in the overall decameric structure.