Thermophilic bacteria are potential sources of novel Rieske non-heme iron oxygenases
Rieske non-heme iron oxygenases (ROs) constitute a large family of oxidoreductase enzymes involved primarily in the oxygenation of various aromatic compounds. Although Gibson et al. (1968) first detected the involvement of such an enzyme system in an alkylbenzene-degrading Pseudomonas sp., the family has since garnered a great deal of attention for two major reasons. First, ROs are key enzymes responsible for the initial attack on otherwise inert aromatic nuclei, thereby making them targets of a cascade of downstream enzymes, leading to their complete mineralization (Gibson and Subramanian 1984; Allen et al. 1995; Gibson and Parales 2000; Mallick et al. 2011). Secondly, regio- and stereoselective cis-dihydroxylation of aromatic compounds, catalyzed by ROs, generate impressive chiral intermediates in the synthesis of a wide array of agrochemically and pharmaceutically important compounds (Ensley et al. 1983; Wackett et al. 1988; Hudlicky et al. 1999; Bui et al. 2002; Newman et al. 2004; Boyd et al. 2005; Zezula and Hudlicky 2005).
Members of the RO family are usually either two- or three-component systems in which one or two soluble electron transport (ET) proteins (such as ferredoxin and reductase) transfer electrons from reduced nucleotides, such as NAD(P)H, to the terminal oxygenase component (a large ?-subunit, often accompanied by a small ?-subunit),which in turn catalyzes the di- or mono-oxygenation of the aromatic nucleus of the substrate (Mason and Cammack 1992; Ferraro et al. 2005). Numerous ROs have been identified and characterized from bacteria, thereby enriching the available information on their diversity in terms of both sequence and function (Habe and Omori 2003; Iwai et al. 2010, 2011; Chakraborty et al. 2012). Although found in all three domains of life, studies have shown that ROs occur more commonly in bacteria compared with archaea and eukaryotes (Chakraborty et al. 2012). Homologs of the large (?) subunit of RO terminal oxygenase (ROox) have also been investigated in certain plant species, such as Arabidopsis thaliana, Zea mays, Pisum sativum, Oryza sativa, Physcomitrella patens, Amaranthus tricolor, Ocimum basilicum and Spinacia oleracea (Caliebe et al. 1997; Meng et al. 2001; Reinbothe et al. 2004; Berim et al. 2014), as well as in insects, nematodes and vertebrates (Rottiers et al. 2006; Yoshiyama et al. 2006; Yoshiyama-Yanagawa et al. 2011). ROs from these taxa, however, have entirely different functions from those of bacterial aromatic ring-hydroxylating ROs. They either act as proteintranslocons, facilitating transport across the chloroplastic envelope membranes during chlorophyll biosynthesis, or are involved in flavone and hormone metabolism in plants. They have also been suggested to be involved in regulation of cholesterol metabolism or trafficking during steroid synthesis in insects (Caliebe et al. 1997; Meng et al. 2001; Reinbothe et al. 2004; Rottiers et al. 2006; Yoshiyama et al. 2006; Yoshiyama-Yanagawa et al. 2011; Berim et al. 2014).
Interestingly, few bacterial RO homologs with novel functions, such as oxidative cyclization during biosynthesis of certain antibiotics, hydroxylation and desaturation of short-chain tertiary alcohols and alkane monooxygenation, have been reported in recent years (Sydor et al. 2011; Schäfer et al. 2012; Li et al. 2013). This suggests that ROs bear much more catalytic potential than previously realized. Almost all bacterial ROs characterized biochemically to date have been isolated from mesophilic bacteria, with the sole exception of polychlorinated biphenyl degrading ring-hydroxylating dioxygenase from Geobacillus sp. JF8 (Mukerjee-Dhar et al. 2005; Shintani et al. 2014). As such, very little is known about RO homologs present in bacteria that live in extreme environments. Extremophiles, and in particular their enzymes, have proved to be a potentially valuable resource in the development of novel biotechnological processes. The most well-studied extremophiles include thermophiles and hyperthermophiles, and enzymes isolated from such microorganisms are often extremely thermostable and resistant to proteolysis, chemical denaturants, detergents, and organic solvents (Vieille and Zeikus 2001). Apart from enzymatic stability at high temperatures, which is often desired in industrial processes, there are several advantages of thermophilic systems in bioremediation studies. Owing to the poor aqueous solubility of aromatic hydrocarbons, biodegradation studies often encounter problems related to bioavailability. These issues can be overcome at elevated temperatures, since bioavailability tends to increase with temperature owing to increases in solubility (Margesin and Schinner 2001; Feitkenhauer and Märk 2003; Perfumo et al. 2007). Thermophilic microorganisms may thus be attractive candidates for sources of novel thermostable ROs with potential utility in industrial biosynthesis and bioremediation at elevated temperatures.
In recent years, microbial genome sequencing projects have generated an enormous quantity of data for public databases. Since publication of the genome sequence of the first extremophile in 1996 (Bult et al. 1996), there has been a substantial increase in the number of extremophilic genome sequences. Metagenomics and single-cell genomics further add to this repertoire (Hedlund et al. 2014). The present study explored all available genome sequences of thermophilic bacteria for the presence of RO homologs and predicted their suitability as novel RO candidates for biotechnological applications.