Acyl CoA oxidase activity from Beauveria bassiana an entomopathogenic fungus

ALCONADA MAGLIANO, TERESA M.; JUÁREZ, M.P

JOURNAL OF BASIC MICROBIOLOGY

Willey Interscience

Lugar: Weinheim; Año: 2006 vol. 46 p. 435 - 443

0233-111X

Beauveria bassiana produces acyl-Co oxidase (ACO) in the P20000 g fraction of glucose and alkanegrown cultures that catalyze the oxidation of acyl-CoAs of different chain length. The activity was measured indirectly over the formation of H2O2 via the oxidative-coupled assay system. ACO activity was assessed spectrophotometrically in the P20000 g fraction of glucose-grown (FS0) and n-alkane grown cultures (FSalk), employing acyl-CoAs of 16 to 24 carbons as substrates. A significant increment in the activity was observed in FSalk as compared to that of controls (FS0) in all conditions tested. Tetracosane-grown cultures showed the highest activity with lignoceroyl-CoA. The reaction conditions were optimized employing lignoceroyl-CoA as substrate. A variable lag phase was observed when the activity was measured as a function of time. In the presence of 3-amino-1,2,4-triazole (AT) to prevent H2O2 consumption by endogenous catalase, the lag phase became shorter and disappeared when AT concentrations were raised from 40 to 200 mM, thus enhancing acyl-CoA oxidation. Enzyme activity reached its maximal value in the presence of 240 µg peroxidase, 0.08% Triton X-100 and 36 µM bovine serum albumin. The apparent Km using lignoceroyl as substrate was estimated 2.5 µM. ACO showed high activity and stability between 30 and 40 °C, as well as between 7.0 and 9.0 pH, for 120 min, being 7.0 the optimum pH. Entomopathogenic fungi have the ability to degrade a series of hydrocarbon structures thatare similar or identical to those of their insect host, utilizing them for energy production and incorporating their degradation products into different cellular components (NAPOLITANO and JUAREZ 1997, CRESPO et al. 2000). The insect cuticle is the first barrier against biological or chemical contact insecticides. Although the major bulk components of the insect cuticle are protein and chitin, a thin film of lipids usually composed of complex mixture of saturated and very long chain compounds, mostly hydrocarbons, free fatty acids, wax esters, and glycerides, is specifically located at the cuticular surface, with the main of function restricting water loss. These lipids also participate in the absorption of chemicals, in the chemical communication processes as well as in the regulation of the activity of microorganisms (BLOMQUIST et al. 1987, JUAREZ 1994). Alkane-growth adaptation enhances the fungal ability to kill its insect host (CRESPO et al. 2002, JUAREZ et al. 2004). After the initial oxidation steps [hydrocarbon → alcohol → very long chain fatty acid (VLCFA)], the first enzyme involved in VLCFA β-oxidation in peroxisomes is the acyl-CoA oxidase (ACO), which donates electrons to molecular oxygen, producing hydrogen peroxide (HAYASHI et al. 1999, WINKLER et al. 2003). The presence of different ACOs provides a mechanism for differential regulation, by a coordinate expression or repression of ACOs, in relation to availability of a particular class of different acyl-CoA chain length (e.g. short-, medium-, long- and very long-chain), tissue type and tissue development in different organisms(HOOKS et al. 1995). The β-oxidation pathway showed certain differences in subcellular distribution:in fungi, the β-oxidation is strictly a peroxisomal process, whereas mammals and plantscontain mitochondrial and peroxisomal fatty acid β-oxidation pathways (HAYASHI et al. 1999, EMANUELSSON et al. 2003). In addition to acyl-CoA oxidase, three enzymes constitute the peroxisomal fatty acid β-oxidation system: enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase. These are immunochemically distinguishable from those of the mitochondrial system. Another difference between both systems is that the acyl- CoA dehydrogenase is the initial enzyme involved in mitochondrial β-oxidation (UEDA et al. 1985). The peroxisomal acyl-CoA oxidase has been reported in higher plants (KIND 1993, WINKLER et al. 2003), animals (VAN VELDHOVEN et al. 1992, THI NGO et al. 2003) and some fungi (KUNAU et al. 1987, WANG and THORPE 1991, LUO et al. 2000). Although present in a large number of organisms, the enzymes isolated from alkane utilizing yeasts (e.g., Candida sp., Yarrowia lipolytica) (WANG and THORPE 1991, LUO et al. 2000) and those from rat liver have received the most attention (VAN VELDHOVEN et al. 1991, 1992, VANHOVE et al. 1991). The yeast Candida tropicalis is an important organism to study peroxisome biogenesis and the localization of peroxisomal proteins, since these organelles are rapidly and abundantly induced following growth on either alkane or fatty acid substrates (UEDA et al. 1985, PICATAGGIO et al. 1991). No information is available on the degradation of very long chain hydrocarbons in biological systems, as well as on the enzymes involved in the hydrocarbon catabolic pathway in filamentous fungi. For this reason, this study will contribute tohelp understanding the metabolic pathways involved in hydrocarbon degradation by entomopathogenic fungi.