Characterization of a Sulfur-regulated Oxygenative Alkylsulfatase from Pseudomonas putida S-313

Abstract

The atsK gene of Pseudomonas putida S-313 was required for growth with alkyl sulfate esters as sulfur source. The AtsK protein was overexpressed in Escherichia coli and purified to homogeneity. Sequence analysis revealed that AtsK was closely related to E. coli taurine dioxygenase (38% amino acid identity). The AtsK protein catalyzed the ␣-ketoglutarate-dependent cleavage of a range of alkyl sulfate esters, with chain lengths ranging from C 4 to C 12 , required oxygen and Fe 2؉ for activity and released succinate, sulfate, and the corresponding aldehyde as products. Enzyme activity was optimal at pH 7 and was strongly stimulated by ascorbate. Unlike most other characterized ␣-ketoglutaratedependent dioxygenases, AtsK accepted a range of ␣-keto acids as co-substrates, including ␣-ketoglutarate (K m 140 M), ␣-ketoadipate, ␣-ketovalerate, and ␣-ketooctanoate. The measured K m values for hexyl sulfate and SDS were 40 and 34 M, respectively. The apparent M r of the purified enzyme of 121,000 was consistent with a homotetrameric structure, which is unusual for this enzyme superfamily, members of which are usually monomeric or dimeric. The properties and amino acid sequence of the AtsK enzyme thus define it as an unusual oxygenolytic alkylsulfatase and a novel member of the ␣-ketoglutarate-dependent dioxygenase family.Bacterial enzymes that cleave aliphatic sulfate esters to release the sulfate moiety have been the subject of considerable study, motivated originally by an awareness of the large scale release of synthetic alkyl sulfate esters into the environment. Due to their amphiphilic properties, long chain aliphatic sulfate esters such as sodium dodecyl sulfate (SDS) are in common use as components of surfactant formulations and are consequently discharged into wastewater. A range of bacterial strains able to degrade aliphatic sulfate esters has been isolated from contaminated sources such as sewage sludge on the basis of their ability to utilize aliphatic sulfate esters as carbon sources for growth (for a review, see Ref. 1). In most cases, degradation of alkyl sulfate esters was found to be initiated by alkylsulfatase enzymes that catalyze the hydrolytic cleavage of the ester bond to liberate inorganic sulfate. The resulting parent alcohol is further degraded (1) or incorporated into cellular lipids (2). Cleavage of the sulfate moiety has been studied in some detail, and several alkylsulfatase enzymes have been purified from cell extracts (3-7).The finding that many isolates from environmental sites that had not been contaminated by detergents also exhibit alkylsulfatase activity (8) suggests that such enzymes may play a role in natural environments as well. Naturally occurring alkyl sulfates include methyl, ethyl, and propyl sulfate in avian eggs (9) and the long chain alkyl sulfates that have been found in membrane structures from unicellular algae (10) and seaweed (11). In aerobic soils, 40 -50% of the total sulfur is present as sulfate esters bound to the soil organic matter (12, 13), although the molecular s...

Bioremediation of naphthalene in water by Sphingomonas paucimobilis using new biodegradable surfactants based on poly (ɛ-caprolactone)

“…SDS was biodegraded efficiently at concentrations below its CMC with a mineralization rate significantly higher than that observed in a micellar phase. It has been shown that several bacteria, especially from the genera Pseudomonas, are able to biodegrade SDS through desulfation and further assimilation (Thomas and White, 1989;Kahnert and Kertesz, 2000). The important decrease in biodegradation rate for SDS, at higher concentration (1.41 g l À1 ) than its CMC, is in agreement with the results obtained by other authors (Abboud et al, 2007).…”

Interconversion of two oxidized forms of taurine/α-ketoglutarate dioxygenase, a non-heme iron hydroxylase: Evidence for bicarbonate binding

“…This Escherichia coli protein is a member of a rapidly expanding enzyme superfamily that utilizes mononuclear Fe(II) active sites to catalyze a diverse range of chemical transformations, usually coupled to the oxidative decarboxylation of an ␣-keto acid (2)(3)(4). Other family members include enzymes that modify protein side chains (5,6), repair alkylation-damaged DNA (7), degrade compounds in the environment (8)(9)(10)(11), and synthesize antibiotics (12)(13)(14), plant metabolites (15,16), or other small molecules (17,18). Most representatives carry out specific hydroxylation reactions, but examples of enzymes catalyzing desaturations, ring closures, and ring expansion reactions have also been documented (19).…”

Succinate Complex Crystal Structures of the α-Ketoglutarate-dependent Dioxygenase AtsK

“…The first reports of this phenomenon involved prolyl hydroxylase, the enzyme whose malfunction underlies scurvy (10 -12) in which the uncoupled turnover results in the metal becoming oxidized in an ascorbic acid-reversible manner (13). Similar ascorbate-reversible uncoupled reactions have been reported for several other enzymes in this family, and ascorbic acid is a widespread component in the buffers used to assay these enzymes to avoid rapid inactivation (1). Assuming that the same mechanism of cosubstrate oxidation occurs for the substrate-free complex as in the catalytic cycle, a highly reactive Fe(IV)ϭO-ferryl species would be formed concomitantly with ␣KG decarboxylation.…”

“…A solution containing the mother liquor and 10 mM Fe(NH 4 ) 2 (SO 4 ) 2 ⅐6H 2 O was added stepwise to the crystals to a total Fe 2ϩ concentration of 2 mM. The crystals were incubated under anaerobic conditions for 36 h. The space group changed upon soaking from P2 1 2 1 2 1 to I2 1 2 1 2 1 .…”

“…The alkylsulfatase AtsK from Pseudomonas putida S-313 is a member of the family of non-heme iron(II), ␣-ketoglutarate-dependent dioxygenases (1,2). This remarkable superfamily is widespread among prokaryotic and eukaryotic organisms, and its members catalyze a broad diversity of energetically demanding biosynthetic and degradative reactions including hydroxylation processes, stereoselective desaturation of inactivated carbon-carbon single bonds, and oxidative ring closure (3).…”