RESUMEN

Cutinases are serine esterases that belong to the α/ß hydrolases superfamily. The natural substrates for these enzymes are cutin and suberin, components of the plant cuticle, the first barrier in the defense system against pathogen invasion. It is well-reported that plant pathogens produce cutinases to facilitate infection. Fusarium verticillioides, one important corn pathogens, is an ascomycete upon which its cutinases are poorly explored. Consequently, the objective of this study was to perform the biochemical characterization of three precursor cutinases (FvCut1, FvCut2, and FvCut3) from F. verticillioides and to obtain structural insights about them. The cutinases were produced in Escherichia coli and purified. FvCut1, FvCut2, and FvCut3 presented optimal temperatures of 20, 40, and 35 °C, and optimal pH of 9, 7, and 8, respectively. Some chemicals stimulated the enzymatic activity. The kinetic parameters revealed that FvCut1 has higher catalytic efficiency (Kcat/Km) in the p-nitrophenyl-butyrate (p-NPB) substrate. Nevertheless, the enzymes were not able to hydrolyze polyethylene terephthalate (PET). Furthermore, the three-dimensional models of these enzymes showed structural differences among them, mainly FvCut1, which presented a narrower opening cleft to access the catalytic site. Therefore, our study contributes to exploring the diversity of fungal cutinases and their potential biotechnological applications.

Asunto(s)

Ascomicetos , Fusarium , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/química , Fusarium/genética

RESUMEN

The 6-oxopurine phosphoribosyltransferase (HPRT, EC 2.4.2.8) from the hyperthermophile Pyrococcus horikoshii was expressed in Escherichia coli and purified. Steady-state kinetic studies indicated that the enzyme is able to use hypoxanthine, guanine and xanthine. The first two substrates showed similar catalytic efficiencies, and xanthine presented a much lower value (around 20 times lower), but the catalytic constant was comparable to that of hypoxanthine. The enzyme was not able to bind to GMP-agarose, but was able to bind the other reverse reaction substrate, inorganic pyrophosphate, with low affinity (K(d) of 4.7+/-0.1 mM). Dynamic light scattering and analytical gel filtration suggested that the enzyme exists as a homohexamer in solution.

Asunto(s)

Proteínas Arqueales/metabolismo , Pentosiltransferasa/metabolismo , Pyrococcus horikoshii/enzimología , Secuencia de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Cromatografía en Gel , Dicroismo Circular , Dimerización , Electroforesis en Gel de Poliacrilamida , Guanina/metabolismo , Guanosina Monofosfato/metabolismo , Hipoxantina/metabolismo , Datos de Secuencia Molecular , Pentosiltransferasa/química , Pentosiltransferasa/genética , Pyrococcus horikoshii/genética , Homología de Secuencia de Aminoácido , Xantina/metabolismo