Direct Ring Fission of Salicylate by a Salicylate 1,2-Dioxygenase Activity from Pseudaminobacter salicylatoxidans

Abstract

In cell extracts of Pseudaminobacter salicylatoxidans strain BN12, an enzymatic activity was detected which converted salicylate in an oxygen-dependent but NAD(P)H-independent reaction to a product with an absorbance maximum at 283 nm. This metabolite was isolated, purified, and identified by mass spectrometry and 1 H and 13 C nuclear magnetic resonance spectroscopy as 2-oxohepta-3,5-dienedioic acid. This metabolite could be formed only by direct ring fission of salicylate by a 1,2-dioxygenase reaction. Cell extracts from P. salicylatoxidans also oxidized 5-aminosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-methylsalicylate, 3-and 5-hydroxysalicylate (gentisate), and 1-hydroxy-2-naphthoate. The dioxygenase was purified and shown to consist of four identical subunits with a molecular weight of about 45,000. The purified enzyme showed higher catalytic constants with gentisate or 1-hydroxy-2-naphthoate than with salicylate. It was therefore concluded that P. salicylatoxidans synthesized a gentisate 1,2-dioxygenase with an extraordinary substrate range, which also allowed the oxidation of salicylate.Naphthalenesulfonic acids are produced on a large scale as industrial detergents, dispersive materials, and intermediates for the production of azo dyes (34). Some pure and mixed bacterial cultures which grow with naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid (2NS), 2-aminonaphthalene-1-sulfonic acid, 6-aminonaphthalene-2-sulfonic acid (6A2NS), or naphthalenedisulfonic acids as the sole source of carbon and energy have been isolated. The initial reaction in the microbial degradation of (substituted) naphthalenesulfonic acids is catalyzed by desulfonating dioxygenases, which catalyze the formation of (substituted) 1,2-dihydroxynaphthalene(s) and the release of sulfite (4, 5, 26-30, 32, 44). Thus, the degradative pathway for naphthalenesulfonic acids converges at the level of 1,2-dihydroxynaphthalene (1,2-DHN) with the well-known pathway for the degradation of naphthalene (Fig. 1). The reactions which are catalyzed by the "upper" naphthalenesulfonic acid pathway result in the formation of (substituted) salicylate(s). For Sphingomonas xenophaga BN6, it was found that the enzymes participating in the upper naphthalenesulfonic acid pathway convert a wide range of substituted substrates and that therefore a single set of enzymes is sufficient for the conversion of various substituted naphthalenesulfonic acids (19-21, 26, 27, 35). In contrast to the degradation of (substituted) naphthalenesulfonic acids to the corresponding salicylates, the productive metabolism of (substituted) salicylates requires rather different metabolic pathways. Thus, salicylate is usually converted by a salicylate 1-monooxygenase to catechol, 3-hydroxysalicylate (2,3-dihyroxybenzoate) is directly oxidized by an extradiol ring fission reaction to a nonaromatic product, 4-hydroxysalicylate (2,4-dihydroxybenzoate) is converted by a monooxygenase to the ring fission substrate 1,2,4-trihydroxybenzene, and gentisate (5-hydroxysalicylate...

Biodegradation of 5-Nitroanthranilic Acid by Bradyrhizobium sp. Strain JS329

“…The properties of the enzyme catalyzing 5NSA oxidation were similar to those of salicylate 1,2-dioxygenase: 1 mol of oxygen was consumed per mol of substrate, 40% to 50% of original activity was lost after 1 week of storage at Ϫ20°C, Fe 2ϩ was required, NAD(P)H was not required, and the enzyme was active against salicylate and inhibited by iron chelators (12).…”

Biodegradation of phenanthrene by Pseudomonas sp. strain PPD: purification and characterization of 1-hydroxy-2-naphthoic acid dioxygenase

“…Gentisic acid 1,2-dioxygenase (GDO), an extradiol-type dioxygenase, has been shown to act on halogenated and alkylated derivatives of gentisic acid but not on salicylic acid or 1-H2NA (Crawford et al, 1975;Feng et al, 1999;Harpel & Lipscomb, 1990;Werwath et al, 1998). Salicylic acid 1,2-dioxygenase (SDO) from Pseudoaminobacter salicylatoxidans showed activity on derivatives of salicylic acid, gentisic acid and 1-H2NA (Hintner et al, 2001(Hintner et al, , 2004. Based on the structure of GDO and SDO, it was proposed that the N-terminal residues, which contribute to the active site, may be responsible for the wide substrate specificity of SDO (Matera et al, 2008).…”

Molecular and Biochemical Characterization of the 5-Nitroanthranilic Acid Degradation Pathway in Bradyrhizobium sp. Strain JS329

“…The mechanism of salicylate dioxygenase-catalyzed elimination of Cl Ϫ from 5-chlorosalicylate is well established, however (13). Salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans catalyzes the oxidative ring cleavage of 5-halosalicylates and produces the corresponding lactone, with the simultaneous release of hydrogen halides (HX), followed by abiotic hydrolysis of the lactone to form maleylpyruvate (12,13).…”