Structural and functional characterization of glutathione transferases isoenzymes

Glutathione transferases (GSTs) are enzymes that normally act as dimeric proteins and catalyze the conjugation of the tripeptide glutathione (GSH) with a number of electrophilic, lipophilic substances creating a water soluble product with reduced toxicity. The overall objective of the present thesis is the study the detoxification role of GSTs enzymes isolated from plants and bacteria. Concerning the bacterial GSTs isoenzymes the study was focused on the genome of Agrobacterium tumefaciens C58. A genome survey revealed the presence of eight GST like proteins in Agrobacterium tumefaciens C58 (AtuGSTs). The substrate specificity of the AtuGST family members was investigated in order to identify catalytic activities that may be related to their biological function. The results showed that AtuGSTs catalyze a broad range of reactions with different members of the family exhibiting quite varied substrate specificity. A GST like sequence from Agrobacterium tumefaciens C58 (AtuGST4) with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct from previously described GST classes. Functional analysis showed that AtuGST4 exhibits significant transferase activities against the common substrates aryl halides as well as very high peroxidation activity towards organic hydroperoxides. The crystal structure of AtuGST4 has been determined at 1.4 Α resolution in complex with S (p-nitrobenzyl) glutathione (Nb GSH). Although AtuGST4 adopts the canonical GST fold we identified sequence and structural characteristics distinct from previously characterized GSTs. The wide catalytic function of this enzyme together with the restricted distribution of this class to soil bacteria may indicate a specific functional role for this enzyme in soil bacteria. Regarding the study of plant GST isoenzymes two GST genes from Glycine max were cloned and expressed in E. coli. The expressed proteins (GmGSTU2-2 and GmGSTU10-10) were efficiently purified (>98% purity) using GSH-agarose affinity chromatography adsorbents. Enzyme activities were measured for different substrates. A study for the inhibition of GST activity of GmGST2-2 and GwGST210-10 was also carried out using a large number of herbicides and insecticides. Another important goal of the present thesis was to develop a rapid and reliable method of screening of GST isoenzymes and mutant libraries. A rapid quantitative screening method for GSTs based on colorimetric measurement of halogen ions released from halogenated xenobiotics was developed. The assay is based on the color formation resulting from the reaction of Hg(SCN)₂ with the released halogen ion of the substrate in the presence of Fe³⁺. The color intensity is proportional to the extent of the catalytic reaction allowing a quantitative measurement of the GST catalytic activity. It was concluded that the proposed colorimetric assay is selective and sensitive and allows the screening of large numbers of samples within a few minutes. The ultimate goal is the application of this method in order to evaluate the biodegradative ability of GSTs to transform halogenated toxic enviionmental pollutants into non toxic products.Οι τρανσφεράσες της γλουταθειόνης (GSTs) είναι ένζυμα που συνήθως δρουν ως διμερή και καταλύουν τη σύζευξη του τριπεπτιδίου γλουταθειόνη (GSΗ) με έναν αριθμό ηλεκτρονιόφιλων, λιπόφιλων ουσιών δημιουργώντας ένα υδατοδιαλυτό προϊόν μειωμένης τοξικότητας. Ο στόχος της παρούσας διδακτορικής διατριβής άφορα κυρίως στη μελέτη του αποτοξινωτικού ρόλου ενζύμων GSTs ο οποίος βασίζεται στην κατάλυση αντιδράσεων σύζευξης με τονικές ενώσεις και η ανάλυση της συμπεριφοράς φυτικών και βακτηριακών GSTs. Κατά την αρχική μελέτη βακτηριακών ισοενζύμων GSTs πραγματοποιήθηκε μελέτη του γονιδιώματος του βακτηριακού στελέχους Agrobacterium tumefaciens C58. Τα ισοένζυμα που απομονώθηκαν και κλωνοποιήθηκαν από το βακτηριακό στέλεχος Agrobacterium tumefaciens C58 εκφράστηκαν ετερόλογα σε κύτταρα E. coli BL21 (DE3). Η σύγκριση της νουκλεοτιδικής αλληλουχίας των ενζύμων επέτρεψε τη φυλογενετική μελέτη τους και τον προσδιορισμό της εξελικτικής σχέσης τόσο μεταξύ τους όσο και με GSTs από άλλους οργανισμούς. Στη συνέχεια πραγματοποιήθηκε μελέτη της ενζυμικής τους δραστικότητας έναντι μεγάλου εύρους υποστρωμάτων. Από το σύνολο των 8 ισοενζύμων που μελετήθηκαν επιλέχθηκε το ισοένζυμο AtuGST4 το οποίο παρουσίασε αξιοσημείωτα κινητικά χαρακτηριστικά προκειμένου να πραγματοποιηθεί περαιτέρω κινητική και δομική μελέτη του ενζύμου. Η τρισδιάστατη δομή του ένζυμου AtuGST4 προσδιορίσθηκε με κρυσταλλογραφία ακτινών-Χ σε σύμπλοκο με τον αναστολέα νιτροβενζυλ-γλουταθείο. Η κρυσταλλική δομή του AtuGST4 προσδιορίστηκε με ανάλυση σε 1.4 Α. Παρόλο που το ένζυμο AtuGST4 υιοθετεί την κανονική GST αναδίπλωση, αναγνωρίστηκαν ξεχωριστά χαρακτηριστικά στη δομή και την αλληλουχία της πρωτεΐνης που τη διαφοροποιούν από τις υπόλοιπες ήδη χαρακτηρισμένες GSTs. Η ευρεία καταλυτική λειτουργική ικανότητα του ενζύμου σε συνδυασμό με την περιορισμένη κατανομή ομολόγων GSTs σε βακτήρια εδάφους ίσως υπαγορεύουν έναν συγκεκριμένο λειτουργικό ρόλο για το ένζυμο AtuGST4 ανάμεσα στα βακτήρια που διαβιούν στο έδαφος. Όσον αναφορά τη μελέτη φυτικών ισοενζύμων GSTs, αυτή περιλαμβάνει δυο ένζυμα (GmGSTU2-2 και GmGSTU10-10) από σόγια (Glycine max) που ανήκουν στην οικογένεια τ (tau) των GSTs. Τα ένζυμα μελετήθηκαν σε κινητικό και μοριακό επίπεδο και προσδιορίστηκαν οι κινητικές τους σταθερές για διαφορετικά υποστρώματα τα βέλτιστα pH δράσης, η επίδραση του ιξώδους στις κινητικές σταθερές, η επίδραση της θερμοκρασίας στην ταχύτητα τα καταλυτικής αντίδρασης καθώς και το θερμοδυναμικό προφίλ των δυο ενζύμων. Ένας ακόμη σημαντικός στόχος της παρούσας διατριβής αφορά την ανάπτυξη μιας ταχείας και αξιόπιστης μεθόδου σάρωσης και ελέγχου μεγάλου αριθμού ισοενζύμων GSTs και μεταλλαγμένων μορφών προκειμένου να καθοριστεί ποιες από αυτές αποτελούν αποτελεσματικούς βιοκαταλύτες σε αντιδράσεις απαλογόνωσης. Η μέθοδος για τον προσδιορισμό της ενζυμικής δραστικότητας βασίστηκε στον σχηματισμό χρωμογόνου (460 nm) που προκύπτει από την αντίδραση ιόντων υδράργυρου με ελεύθερα ιόντα αλογόνων που προκύπτουν από την απαλογόνωση των υποστρωμάτων. Επιπροσθέτως, η ένταση του χρώματος δείχνει τον βαθμό την καταλυτικής δράσης των ενζύμων που μας επιτρέπει να επιλέξουμε όχι μόνο τους θετικούς κλώνους αλλά και εκείνους με την υψηλότερη καταλυτική ικανότητα. Η μέθοδος ανίχνευσης αλογονοιόντων που αναπτύχθηκε στην παρούσα διατριβή χρησιμοποιήθηκε για τη μελέτη του ενζύμου GmGSTU4-4 (από Glycine max) και τη σάρωση μεταλλαγμένων μορφών του ενζύμου που προέκυψαν από DNA shuffling ως προς διάφορα υποστρώματα και πιθανούς αναστολείς

Fungal-bacterial “deposit” into the oil degradation “bank”

Several reports have been published about the broad distribution of petroleum-degrading microorganisms in soil, water and sediments but they may not be highly effective or be present in sufficient numbers to accomplish contaminant remediation [Yakimov et al (2007) Curr. Opinion in Biotechnol.18: 257–266, Joo et al(2008) Env. Pol. 156: 891–896]. The current study investigates the biodegradation of polycyclic aromatic hydrocarbons (PAHs) by both autochthonous microorganisms isolated from long-term oil contaminated environments and known fungi-bacteria consortia with proved biodegradation abilities. Potential PAHs degraders have also been sought in extreme environmental conditions such as exceptional salinity or temperature. Liquid and soil environmental samples were used to isolate the oil-degrading microorganisms and their oil removal efficiency will be evaluated in liquid cultures enriched with diesel or mixed PAHs (C6-C9). The next experimental steps will be focused on the molecular analyses of microbial communities in order to identify key species, the characterization of the isolated strains in terms of degradation potential and finally the establishment and characterization of fungal-bacteria consortia.The enzymes involved in the biodegradation process will be investigated for the best performing degraders,in order to clarify the relationship between their bioremediation potency and enzymatic activity. Combining the knowledge obtained from the whole cell and enzymatic “identities” of these consortia, the challenge is to obtain high bioremediation rates, in the real field, surpassing the barriers of a time- and cost-consuming treatment

Steady-state kinetic analysis of <i>Atu</i>GSTH1-1.

a<p>k_cat values were calculated for the substrates CDNB, CuOOH, and HEDS.</p

Kinetic parameters of mutant enzymes for the CDNB/GSH and CuOOH/GSH reactions catalyzed by <i>Atu</i>GSTH1-1.

a<p>ND: Non determined.</p

Close-up stereo view of the active site. Hydrogen-bonds (<4.0 Å) between Nb-GSH and the enzyme are shown as dashed lines.

<p>W304 and W117 from the proposed electron-sharing network are depicted. The orientation of Nb-GSH is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034263#pone-0034263-g003" target="_blank">Figure 3C</a>. The figure was produced using the CCP4 molecular graphics program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034263#pone.0034263-Pterton1" target="_blank">[56]</a>.</p

The effect of viscosity on k_cat for the CDNB/GSH and CuOOH/GSH reactions catalyzed by <i>Atu</i>GSTH1-1 and its mutants.

<p>The slopes for the wild-type and the mutant enzymes were derived from the linear plot of the relative turnover number (k^o_cat/k_cat) as a function of relative viscosity (η/η^o) using glycerol as co-solvent.</p

Data collection and refinement statistics.

#<p>Numbers in parenthesis correspond to the highest resolution shell.</p>&<p>Redundancy-independent R-value <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034263#pone.0034263-Diederichs1" target="_blank">[54]</a>.</p

Crystal structure of <i>Atu</i>GSTH1-1.

<p><b>A.</b> Ribbon diagram of <i>Atu</i>GSTH1-1 monomer. Assignment of secondary structure elements was carried out by DSSP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034263#pone.0034263-Kabsch2" target="_blank">[55]</a>. The bound Nb-GSH and phosphate ion are shown as sticks coloured according to atom type. <b>B.</b> Ribbon diagram of the dimeric <i>Atu</i>GSTH1-1 structure. The 2-fold axis is perpendicular to the plane of the page. Subunit A is colored in cyan and subunit B is in lemon yellow. The inhibitor Nb-GSH is represented with sticks and coloured according to atom type. <b>C.</b> Stereo view of the mFo-DFc electron density omit map for the bound Nb-GSH contoured at 3σ. The figures were produced using the CCP4 molecular graphics program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034263#pone.0034263-Pterton1" target="_blank">[56]</a>.</p

Multiple sequence alignment of <i>Atu</i>GST4 with representative GST sequences.

<p>Alpha, (GSTA, AAA16572); beta, (GSTB, CAR42930); delta, (GSTD, EDW42478); epsilon, (GSTE, EDV55071); phi, (GSTF, AAA33469); kappa, (GSTK, EDM15501); lambda, (GSTL, BT051648); mu, (GSTM, AAC17866); omega, (GSTO, EDL42044); pi, (GSTP, AAP72967); ro, (GSTR, ABV24478); sigma, (GSTS, EAA45010); theta, (GSTT, BAB39498); tau, (GSTU, ABF99228), chi, (GSTX, EAW33767); and zeta, (GSTZ, P28342). NCBI accession numbers are in parentheses. The degree of conservation is shown below the alignments in green. Amino acids are colored according to polarity or charge (red for negative charged, blue for positive charged, black for neutral and green for uncharged polar amino acids).</p

A glutathione transferase from Agrobacterium tumefaciens reveals a novel class of bacterial GST superfamily.

In the present work, we report a novel class of glutathione transferases (GSTs) originated from the pathogenic soil bacterium Agrobacterium tumefaciens C58, with structural and catalytic properties not observed previously in prokaryotic and eukaryotic GST isoenzymes. A GST-like sequence from A. tumefaciens C58 (Atu3701) with low similarity to other characterized GST family of enzymes was identified. Phylogenetic analysis showed that it belongs to a distinct GST class not previously described and restricted only in soil bacteria, called the Eta class (H). This enzyme (designated as AtuGSTH1-1) was cloned and expressed in E. coli and its structural and catalytic properties were investigated. Functional analysis showed that AtuGSTH1-1 exhibits significant transferase activity against the common substrates aryl halides, as well as very high peroxidase activity towards organic hydroperoxides. The crystal structure of AtuGSTH1-1 was determined at 1.4 Å resolution in complex with S-(p-nitrobenzyl)-glutathione (Nb-GSH). Although AtuGSTH1-1 adopts the canonical GST fold, sequence and structural characteristics distinct from previously characterized GSTs were identified. The absence of the classic catalytic essential residues (Tyr, Ser, Cys) distinguishes AtuGSTH1-1 from all other cytosolic GSTs of known structure and function. Site-directed mutagenesis showed that instead of the classic catalytic residues, an Arg residue (Arg34), an electron-sharing network, and a bridge of a network of water molecules may form the basis of the catalytic mechanism. Comparative sequence analysis, structural information, and site-directed mutagenesis in combination with kinetic analysis showed that Phe22, Ser25, and Arg187 are additional important residues for the enzyme's catalytic efficiency and specificity