Title
A Sulfur Oxygenase from the Haloalkaliphilic Bacterium Thioalkalivibrio paradoxus with atypically low Reductase Activity
Author
Patrick Rühl, Uwe Pöll, Johannes Braun, Andreas Klingl, Arnulf Kletzin
Year
2016
Journal
Journal of Bacteriology
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 (29-34% identity). A synthetic gene encoding the TpSOR expressed in E. coli resulted in a protein of 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 10-98°C (Topt≈80°C) and a pH range of 6-11.5 (pHopt≈9; 308±78 U/mg protein). Sulfide formation had a maximum specific activity of 0.03 U/mg, or less than 1% of the corresponding activity of other SORs. Hence, reductase activity seems not to be an integral part of the reaction mechanism. The TpSOR was most active at NaCl or glycine betaine concentrations of 0-1 M although 0.2% of the maximal activity was detected even at 5 M NaCl and 4 M betaine. The melting point of the TpSOR was close to 80°C, when monitored by CD spectroscopy or differential scanning fluorimetry, however denaturation kinetics were slow: 55% of the residual activity remained after 25 min incubation at 80°C. Site-directed mutagenesis showed that the active-site residue Cys44 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.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Thermal stability, Biochemistry