Characterization of the enzymes involved in the diolsynthase pathway in Pseudomonas aeruginosa

Abstract

In 2015 our research group described, in P. aeruginosa, the oleate-diol synthase system which consists of two sequentially and independently enzymatic reactions: 10S-Dioxygenase (10-DOX) (PA2077) used oleic acid (as preferred substrate) and converted into hydroperoxide 10-H(P)OME ((10S)-hydroxy(per)oxi-(8E)- octadecenoic acid), followed by the bioconversion of the hydroperoxide into 7,10- DiHOME ((7S, 10S)-dihydroxy-(8E)-octadecenoic acid) by an 7,10-diol synthase (7,10-DS) (PA2078). This products, synthetized in the periplasm, are specifically exported though ExFadLO outer-membrane transport to the extracellular medium, where they accumulated and act as bioactive compounds. Finally a new single operon is responsible for this diol synthase activity that contains two genes PA2077 and PA2078 contiguously located in the genome.

In this study we characterized recombinant 10S-DOX and 7,10-DS enzymes, expressed by E. coli and demonstrated that other proteobacteria have ability to produce oxylipins. Also we have shown that E. coli DH5α/pMMB-77 and E. coli BL21/pET28a-78 can express aggregate proteins as inclusion bodies under stress conditions with catalytic activity consist of the corresponding enzymes.

As accessing to commercial 10-H(P)OME is not available, for the first time produced 10-H(P)OME from P. putida (pBBR-77) by 10S-DOX during 1 h of incubation time in the presence of oleic acid as substrate at 30 ºC. Biochemical characterization of soluble 10S-DOX and 7,10-DS have shown, maximum enzyme activity for 10S-DOX was at 30 ºC and for 7,10-DS at 35 ºC. Also,

results indicated the thermal stability of 7,10-DS was slightly higher than of 10S- DOX, but both enzymes losing most of their activates at 50 ºC (only remained 20% of activity). Activity of 10S-DOX and 7,10-DS in different range of pH 7-10 show activity of both of them increasing between this range of pH. The effect divalent metal ions (1 mM) on 10S-DOX and 7,10-DS was assayed. The activity of 7,10-DS increased in the presence of Mg2+, Cd2+, and Ni2+ while the activity of 10S-DOX was decreased in the presence of same metal ions. After inducing recombinant E. coli DH5α/pMMB-77 and E. coli BL21/pET28a-78 by IPTG, insoluble IBs were obtained to purification and refolding. Study of functional activity of purified IBs have shown in both cases enzymes were trapped in IBs-77 and IBs-78 with activity. Structure and morphology of IBs were characterized by different techniques. Transmission electron microscopy (TEM), demonstrated size and morphology of IBs. Images have shown IBs-77 and IBs-78 appears as dark and dense region inside cells that it seems IBs-78 are denser than IBs-77. Infrared spectrometry (FTIR) and dye binding with Congo red (CR) and Thioflavin T (ThT) were used to reveal the amyloid structure of IBs. In both cases, the spectra obtained from FTIR displayed amyloid aggregate which consist of β-sheet structure. Also dye binding demonstrated presence of amyloid structure in IBs-77 and IBs-78. Atomic force microscopy (AFM) provide direct images of IBs before and after digestion with Proteinase K that were observed purified IBs-77 collapse about 61- 65% after digestion and 70-84% in the case of IBs-78. In order to assess whether the diol synthase system is a common occurrence among bacteria, an experimental screening of other proteobacteria demonstrated that some of the species belong to the genera Aeromonas, Pseudoalteromonas, Shewanella, Thauera, Ensifer and Pseudomonas were able to produce 10S-H(P)OME and 7,10- DiHOME from OA as substrate, which are the same products obtained from P. aeruginosa diol synthase pathway.

Els objectius de la present Tesi Doctoral titulada són: Caracterització dels enzims de la via oleat diol-sintasa descrita en Pseudomonas aeruginosa. Producció i estudi funcional, estructural i morfològic dels cossos d’inclusió dels enzims de la via de la oleat diol-sintasa. Rastreig experimental de la 10S-DOX i lla 7,10-DS en diversos proteobacteris.

Recentment s’ha descrit que aquesta via consta de dos passos; el primer enzim, la 10S-dioxygenasa (10S-DOX ) catalitza la formació de l’àcid 10S-hydroperoxy- 8E-octadecenoic (10SH(P)HOME) a partir de l’àcid oleic i un segon enzim la 7S,10S-diol sintasa (7,10-DS) catalitza la formació de l’àcid 7S,10S-dihidroxi-8E- octadecenoic (7S,10S-DiHOME) a partir del 10S-H(P)HOME. Ambdós enzims contenen un mol of Fe+2 lligat al grup hemo com a grup prostètic, propietat característica de la subfamília FadCCPs. A partir de l’expressió heteròloga de la 10S-DOX i de la 7,10-DS s’ha procedit a la seva caracterització bioquímica. Les constants cinètiques per 10S-DOX han estat: Km, 0.84 mM i Vmax de 13.9 Ug-1 min-1 ; en el cas de la 7,10-DS el valor de la Km es de 1,84 mM i la Vmax de 35,17 Ug-1 min-1 . Ambdós enzims presenten activitats similars en el rang de pH 7-10 i una temperatura òptima de 30 °C i de 35 °C per la 10S-DOX i la 7,10-DS respectivament. La presència de cations divalents inhibeix l’activitat de la 10S-DOX, mentre que Ni2+, Mg2+ i Cd2+ augmenten l’activitat de la 7,10-DS. Els resultats obtinguts del rastreig experimental amb diversos proteobacteris seleccionats d’acord al seu metabolisme i habitat, indiquen que la presència d’un putatiu sistema de la diol-sintasa no està restringit a Pseudomonas aeruginosa. En la sobreexpressió d’ ambdós enzims, en determinades soques d’ E. coli, s’obtenen les proteïnes agregades formant cossos d’inclusió (IBs) que es localitzen majoritàriament en els pols de les cèl·lules. En els dos casos 10S-DOX i 7,10-DS presenten activitat catalítica. L’estudi estructural mitjançant FT-IR, Vermell Congo i fluorescència (Tiamina T) indiquen que en els dos casos tenen estructura amiloide. Tant els cossos d’inclusió de la 10S-DOX com en el cas de la 7,10-DOX son sensibles al digestió amb proteïnasa K, la qual provoca un col·lapse de l’ordre del 61-65% del cossos d’inclusió de la 10S-DOX i del 70-84% en el cas dels cossos d’inclusió de la 7,10-DS.