Michael A. Kertesz

Riding the sulfur cycle ^ metabolism of sulfonates and sulfate esters in Gram-negative bacteria

Sulfonates and sulfate esters are widespread in nature, and make up over 95% of the sulfur content of most aerobic soils. Many microorganisms can use sulfonates and sulfate esters as a source of sulfur for growth, even when they are unable to metabolize the carbon skeleton of the compounds. In these organisms, expression of sulfatases and sulfonatases is repressed in the presence of sulfate, in a process mediated by the LysR-type regulator protein CysB, and the corresponding genes therefore constitute an extension of the cys regulon. Additional regulator proteins required for sulfonate desulfonation have been identified in Escherichia coli (the Cbl protein) and Pseudomonas putida (the AsfR protein). Desulfonation of aromatic and aliphatic sulfonates as sulfur sources by aerobic bacteria is oxygen-dependent, carried out by the alpha-ketoglutarate-dependent taurine dioxygenase, or by one of several FMNH(2)-dependent monooxygenases. Desulfurization of condensed thiophenes is also FMNH(2)-dependent, both in the rhodococci and in two Gram-negative species. Bacterial utilization of aromatic sulfate esters is catalyzed by arylsulfatases, most of which are related to human lysosomal sulfatases and contain an active-site formylglycine group that is generated post-translationally. Sulfate-regulated alkylsulfatases, by contrast, are less well characterized. Our increasing knowledge of the sulfur-regulated metabolism of organosulfur compounds suggests applications in practical fields such as biodesulfurization, bioremediation, and optimization of crop sulfur nutrition.

Characterization of alpha-ketoglutarate-dependent taurine dioxygenase from Escherichia coli.

The Escherichia coli tauD gene is required for the utilization of taurine (2-aminoethanesulfonic acid) as a sulfur source and is expressed only under conditions of sulfate starvation. The sequence relatedness of the TauD protein to the α-ketoglutarate-dependent 2,4-dichlorophenoxyacetate dioxygenase of Alcaligenes eutrophus suggested that TauD is an α-ketoglutarate-dependent dioxygenase catalyzing the oxygenolytic release of sulfite from taurine (van der Ploeg, J. R., Weiss, M. A., Saller, E., Nashimoto, H., Saito, N., Kertesz, M. A., and Leisinger, T. (1996) J. Bacteriol. 178, 5438–5446). TauD was overexpressed in E. coli to ∼70% of the total soluble protein and purified to apparent homogeneity by a simple two-step procedure. The apparent M r of 81,000 of the native protein and the subunit M rof 37,400 were consistent with a homodimeric structure. The pure enzyme converted taurine to sulfite and aminoacetaldehyde, which was identified by high pressure liquid chromatography after enzymatic conversion to ethanolamine. The reaction also consumed equimolar amounts of oxygen and α-ketoglutarate; ferrous iron was absolutely required for activity; and ascorbate stimulated the reaction. The properties and amino acid sequence of this enzyme thus define it as a new member of the α-ketoglutarate-dependent dioxygenase family. The pure enzyme showed maximal activity at pH 6.9 and retained activity on storage at −20 °C for several weeks. Taurine (K m = 55 μm) was the preferred substrate, but pentanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, and 1,3-dioxo-2-isoindolineethanesulfonic acid were also desulfonated at significant rates. Among the cosubstrates tested, only α-ketoglutarate (K m = 11 μm) supported significant dioxygenase activity.

Posttranslational Formation of Formylglycine in Prokaryotic Sulfatases by Modification of Either Cysteine or Serine

Eukaryotic sulfatases carry an α-formylglycine residue that is essential for activity and is located within the catalytic site. This formylglycine is generated by posttranslational modification of a conserved cysteine residue. The arylsulfatase gene ofPseudomonas aeruginosa also encodes a cysteine at the critical position. This protein could be expressed in active form in a sulfatase-deficient strain of P. aeruginosa, thereby restoring growth on aromatic sulfates as sole sulfur source, and inEscherichia coli. Analysis of the mature protein expressed in E. coli revealed the presence of formylglycine at the expected position, showing that the cysteine is also converted to formylglycine in a prokaryotic sulfatase. Substituting the relevant cysteine by a serine codon in the P. aeruginosa gene led to expression of inactive sulfatase protein, lacking the formylglycine. The machinery catalyzing the modification of thePseudomonas sulfatase in E. coli therefore resembles the eukaryotic machinery, accepting cysteine but not serine as a modification substrate. By contrast, in the arylsulfatase ofKlebsiella pneumoniae a formylglycine is found generated by modification of a serine residue. The expression of both theKlebsiella and the Pseudomonas sulfatases as active enzymes in E. coli suggests that two modification systems are present, or that a common modification system is modulated by a cofactor.

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

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 α-ketoglutarate-dependent cleavage of a range of alkyl sulfate esters, with chain lengths ranging from C4 to C12, required oxygen and Fe2+ 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 α-ketoglutarate-dependent dioxygenases, AtsK accepted a range of α-keto acids as co-substrates, including α-ketoglutarate (K m 140 μm), α-ketoadipate, α-ketovalerate, and α-ketooctanoate. The measuredK m values for hexyl sulfate and SDS were 40 and 34 μm, respectively. The apparent M rof 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 α-ketoglutarate-dependent dioxygenase family.

Genetic organization of sulphur‐controlled aryl desulphonation in Pseudomonas putida S‐313

Pseudomonas putida S‐313 is able to desulphonate a broad range of aromatic sulphonates to provide sulphur for growth by monooxygenolytic cleavage to yield the corresponding phenol. After miniTn5 transposon mutagenesis of this strain, 11 mutants were isolated that were no longer able to utilize benzenesulphonate as a sulphur source. Three of these mutants were defective in the utilization of all aromatic sulphonates tested, but they grew normally with other sulphur sources. These strains contained independent insertions in the novel 4.2 kb asfRABC gene cluster, encoding a putative reductase (AsfA), a ferredoxin (AsfB), a putative periplasmic binding protein (AsfC), which was localized to the periplasm using alkaline phosphatase fusions, and a divergently oriented fourth gene, asfR, that encoded a LysR‐type regulator protein. A further mutant was interrupted in the ssu locus, which includes the gene for a putative desulphonative monooxygenase. Transformation of Pseudomonas aeruginosa with the asfRAB genes was sufficient to allow arylsulphonate utilization by this species, which does not normally use these compounds, suggesting that the AsfAB proteins may constitute an arylsulphonate‐specific electron transport system that interacts with a less specific oxygenase. Expression of the asfABC genes in P. putida was induced by benzenesulphonate or toluenesulphonate, and it was repressed in the presence of sulphate in the growth medium. AsfR was a negative regulator of asfABC expression, and toluenesulphonate induced expression of these genes indirectly by reducing the expression of the asfR gene.