The Km for the substrate 1-H2NA remained unaltered

in the presence of NADPH or NADH (Table 5). The enzyme showed similar Km for NADPH and NADH (Table 5). The saturation plot for FAD was hyperbolic and Km was determined to be 4.7 μM (Table 5). Alcaligenes sp. strain PPH degrades phenanthrene, hydroxybenzoates (o-, m- and p-) and o-phthalate (Deveryshetty et al., 2007). Based on metabolic analysis, the proposed pathway for phenanthrene degradation is: phenanthrene 1-H2NA 1,2-DHN salicylaldehyde salicylic acid catechol. The steps involved in the metabolism of 1-H2NA to salicylic acid are similar to that involved in naphthalene degradation and hence referred to as the ‘naphthalene Selleckchem Ipatasertib route’. The generated catechol enters the central carbon cycle via the meta ring-cleavage pathway. Organisms capable of degrading phenanthrene via the ‘naphthalene route’ have the ability to degrade naphthalene (Davies & Evans, 1964; Evans et al., 1965; Menn et al., 1993; Sanseverino et al., 1993; Kiyohara et al., 1994; Takizawa et al., 1994; Yang et al., 1994). Interestingly, strain PPH failed to metabolize naphthalene as the carbon source; this could be due to lack of naphthalene dioxygenase or the presence of highly specific phenanthrene dioxygenase in this strain. Compared to salicylate,

phenanthrene-grown cells showed higher specific activity of 1-hydroxy-2-naphthoic acid hydroxylase (Table 2). As observed for several aromatic degradative pathways (Grund et al., 1990; Gescher et al., 2002; Phale et al., 2007; Swetha et al., MK-8669 2007; Deveryshetty & Phale, 2009), enzymes of phenanthrene

degradation in strain PPH were also found to be inducible in nature. The upper-pathway enzymes of naphthalene degradation (naphthalene to salicylic acid) have been proposed to be involved in the conversion of phenanthrene to 1-H2NA and anthracene to 2-hydroxy-1-naphthoic acid (Menn et al., 1993). Further, 1-H2NA was metabolized to 1,2-DHN by salicylate-1-hydroxylase and reported to have broad substrate specificity (Balashova et al., 2001). In Alcaligenes sp. strain PPH, enzyme induction pattern and heat stability studies suggested the existence of two different enzymes, 1-hydroxy-2-naphthoic acid hydroxylase Ribose-5-phosphate isomerase and salicylate-1-hydroxylase, responsible for the conversion of 1-H2NA to 1,2-DHN and salicylic acid to catechol, respectively. The enzyme responsible for the hydroxylation of 1-H2NA has not been reported so far. This is the first study reporting the existence of 1-hydroxy-2-naphthoic acid hydroxylase. The property of heat stability helped to resolve 1-hydroxy-2-naphthoic acid hydroxylase from salicylate hydroxylase and was exploited to partially purify the protein (Table 3). The enzyme was yellow in color and showed characteristic flavoprotein absorption spectrum (Fig. 2), as observed for several other hydroxylases (Yamamoto et al., 1965; Hesp & Calvin, 1969; White-Stevens & Kamin, 1972).