A Sulfur Oxygenase from the Haloalkaliphilic Bacterium Thioalkalivibrio paradoxus with Atypically Low Reductase Activity

Abstract

Sequence comparisons showed that the sulfur oxygenase reductase (SOR) of the haloalkaliphilic bacterium Thioalkalivibrio paradoxus Arh 1 (TpSOR) is branching deeply within dendrograms of these proteins (29 to 34% identity). A synthetic gene encoding TpSOR expressed in Escherichia coli resulted in a protein 14.7 Ϯ 0.9 nm in diameter and an apparent molecular mass of 556 kDa. Sulfite and thiosulfate were formed from elemental sulfur in a temperature range of 10 to 98°C (optimum temperature Ϸ 80°C) and a pH range of 6 to 11.5 (optimum pH Ϸ 9; 308 Ϯ 78 U/mg of protein). Sulfide formation had a maximum specific activity of 0.03 U/mg, or Ͻ1% of the corresponding activity of other SORs. Hence, reductase activity seems not to be an integral part of the reaction mechanism. TpSOR was most active at NaCl or glycine betaine concentrations of 0 to 1 M, although 0.2% of the maximal activity was detected even at 5 M NaCl and 4 M betaine. The melting point of TpSOR was close to 80°C, when monitored by circular dichroism spectroscopy or differential scanning fluorimetry; however, the denaturation kinetics were slow: 55% of the residual activity remained after 25 min of incubation at 80°C. Sitedirected mutagenesis showed that the active-site residue Cys 44 is essential for activity, whereas alanine mutants of the two other conserved cysteines retained about 0.5% residual activity. A model of the sulfur metabolism in T. paradoxus is discussed.IMPORTANCE Sulfur oxygenase reductases (SORs) are the only enzymes catalyzing an oxygen-dependent disproportionation of elemental sulfur and/or polysulfides to sulfite, thiosulfate, and hydrogen sulfide. SORs are known from mesophilic and extremophilic archaea and bacteria. All SORs seem to form highly thermostable 24-subunit hollow spheres. They carry a low-potential mononuclear nonheme iron in the active site and an indispensable cysteine; however, their exact reaction mechanisms are unknown. Typically, the reductase activity of SORs is in the range of 5 to 50% of the oxygenase activity, but mutagenesis studies had so far failed to identify residues crucial for the reductase reaction. We describe here the first SOR, which is almost devoid of the reductase reaction and which comes from a haloalkaliphilic bacterium.KEYWORDS Thioalkalivibrio, alkaliphile, extremophiles, sulfur disproportionation, sulfur oxygenase reductase, thermostability, Allochromatium vinosum, Acidianus ambivalens S ulfur oxygenase reductases (SORs) catalyze a dioxygen-dependent disproportionation reaction of elemental sulfur (S 0 , consisting mostly of cyclo-octasulfur) with sulfite, thiosulfate, and sulfide as detectable products (1-3). It is not yet known whether thiosulfate is a primary reaction product of the enzyme reaction or whether it is formed

A novel bacterial sulfur oxidation pathway provides a new link between the cycles of organic and inorganic sulfur compounds

“…7a, b) can (but does not have to) be combined with the genetic equipment for sulfide detoxification involving Sqr, Rhd, and Pdo [52] (Fig. 7c) and/or cytoplasmically located sulfur oxygenase reductase (SOR), an enzyme that catalyzes oxygen-dependent disproportionation of elemental sulfur and/or polysulfides [53] (Fig. 7d).…”

“…These enzymes usually catalyze a dioxygen-dependent disproportionation of elemental sulfur/and or poylsulfides to hydrogensulfite, thiosulfate, and hydrogen sulfide. Here, SOR is depicted without reductase activity as has been described for Thioalkalivibrio paradoxus [53] TVNIR-0860-0847, pdo: TVNIR_2193, sor: TVNIR_2721 and TVNIR_1174).…”

Persulfide Dioxygenase From Acidithiobacillus caldus: Variable Roles of Cysteine Residues and Hydrogen Bond Networks of the Active Site

“…Sulfite was the only reaction product up to 45°C. At 50°C and above, thiosulfate was also detected, which is the product of a non-enzymatic reaction of sulfite and excess sulfur at elevated temperature (Supplementary Figure S4 ; Roy and Trudinger, 1970 ; Kletzin, 1989 ; Rühl et al, 2017 ). The temperature span of Ac PDO activity was 90 K ( Figure 2A ; note that the activities in the temperature and pH curves were measured at the sub-optimal GSH concentration of 0.2 mM because this had been done before the final optimization of the enzyme assay was accomplished: in this case the total incubation time of the enzyme assay was 10 min with intervals of 2 min).…”

Crystallographic and cryogenic electron microscopic structures and enzymatic characterization of sulfur oxygenase reductase fromSulfurisphaera tokodaii

“…2B). The optimum temperatures of SORs from other thermophiles were reported to be 70-85°C (Kletzin, 1989;Pelletier et al, 2008;Rühl et al, 2017;Sun et al, 2003;Veith et al, 2012). The optimum pH of StSOR was 6.0 for both oxygenase and reductase activities, and oxygenase activity significantly dropped at pH levels above 6.5 (Fig.…”

“…One of the key enzymes in these processes is sulfur oxygenase reductase (SOR,EC 1.13.11.55). SORs are present in a part of thermophilic and mesophilic archaea and in bacteria such as Acidianus ambivalens (AaSOR) (Kletzin, 1989;Urich et al, 2004), Acidianus tengchongensis (AtSOR) (Chen et al, 2005;Sun et al, 2003), Aquifex aeolicus (AqSOR) (Pelletier et al, 2008), Halothiobacillus neapolitanus (HnSOR) (Veith et al, 2012), Sulfobacillus thermosulfidooxidans (SbSOR) (Janosch et al, 2015), and Thioalkalivibrio paradoxus (TpSOR) (Rühl et al, 2017). SOR catalyzes the oxygendependent sulfur oxygenation and disproportionation (both oxidation and reduction) reactions to produce sulfite and hydrogen sulfide and thiosulfate is concomitantly produced by a non-enzymatic reaction:…”