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Rühl, Patrick; Pöll, Uwe; Braun, Johannes; Klingl, Andreas; Kletzin, Arnulf (2017): A Sulfur Oxygenase from the Haloalkaliphilic Bacterium Thioalkalivibrio paradoxus with Atypically Low Reductase Activity. In: Journal of Bacteriology, Vol. 199, No. 4, e00675
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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 +/- 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 degrees C ( optimum temperature approximate to 80 degrees C) and a pH range of 6 to 11.5 ( optimum pH approximate to 9;308 +/- 78 U/mg of protein). Sulfide formation had a maximum specific activity of 0.03 U/mg, or <1% 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 degrees 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 degrees 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 24subunit 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.