Cytosolic Mercaptopyruvate Sulfurtransferase Is Evolutionarily Related to Mitochondrial Rhodanese.

Abstract

Rat liver mercaptopyruvate sulfurtransferase (MST) was purified to homogeneity. MST is very similar to rhodanese in physicochemical properties. Further, rhodanese cross-reacts with anti-MST antibody. Both purified authentic MST and expressed rhodanese possess MST and rhodanese activities, although the ratio of rhodanese to MST activity is low in MST and high in rhodanese. In order to compare the active site regions of MST and rhodanese, the primary structure of a possible active site region of MST was determined. The sequence showed 66% homology with that of rat liver rhodanese. An active site cysteine residue (Cys246; site of formation of persulfide in catalysis) and an arginine residue (Arg185; substrate binding site) in rhodanese were also conserved in MST. On the other hand, two other active site residues (Arg247 and Lys248) were replaced by Gly and Ser, respectively. Conversion of rhodanese to MST was tried by site-directed mutagenesis. After cloning of rat liver rhodanese, recombinant wild type and three mutants (Arg247-->Gly and/or Lys248-->Ser) were constructed. The enzymes were expressed in Escherichia coli strain BL21 (DE3) with a T7 promoter system. The mutation of these residues decreases rhodanese activity and increases MST activity.

Enzymatic Activity of the Arabidopsis Sulfurtransferase Resides in the C-Terminal Domain But Is Boosted by the N-Terminal Domain and the Linker Peptide in the Full- Length Enzyme

“…Both substrates could be metabolized naturally, but the physiological levels of 3-MP are rather low in comparison to the K m determined (Papenbrock and Schmidt, 2000b); the kinetic data may therefore indicate that better substrates still need to be found. The k cat values of the Arabidopsis ST1 were also lower than those determined for STs from other organisms (Westley, 1973;Alexander and Volini, 1987;Nagahara et al, 1995Nagahara et al, , 1999Ray et al, 2000) which might again indicate a divergent substrate specificity. From the point mutant studies C332 was proven to be an essential residue with both substrates used.…”

“…MST is supposed to transfer the sulfur in a single displacement from 3-MP to cyanide as acceptor whereas TST was suggested to follow a double displacement mechanism (Nagahara et al, 1995(Nagahara et al, , 1999. The Arabidopsis protein obviously accepts both substrates and requires C332 to process both, however, the mechanism has not been clarified by our experiments.…”

“…The MST protein is located in the cytosol and in the mitochondria of the eukaryotic cell (Wood and Fiedler, 1953;Meister et al, 1954;Westley, 1973;Nagahara et al, 1999). The enzyme was purified to homogeneity (Nagahara et al, 1995) and cloned from rat liver (Nagahara and Nishino, 1996). Both ST proteins, MST and rhodanese, isolated from the same organism (Rattus) accepted 3-MP and thiosulfate, but the ratios of their respective enzyme activities differed.…”

Evidence for the physiological role of a rhodanese-like protein for the biosynthesis of the molybdenum cofactor in humans

“…In vitro, sulfurtransferase activity can be measured as thiosulfate sulfurtransferase activity by using thiosulfate as sulfur donor or as mercaptopyruvate sulfurtransferase (MST) activity by using ␤-mercaptopyruvate as sulfur donor (20). Cyanide can be used as sulfur acceptor in vitro, yielding thiocyanate (rhodanide) and sulfite or pyruvate, respectively.…”

Intracellular Localization of Arabidopsis Sulfurtransferases

“…The 3-mercaptopyruvate Str protein was localized by immunogold-labeling and western-blot analysis to the cytoplasm and the mitochondria, whereas thiosulfate Str was detected exclusively in mitochondria and mainly in liver cells. 3-mercaptopyruvate Str might detoxify cyanide in the cytoplasm, and in the mitochondria both Str enzymes would protect cytochrome c oxidase effectively (Nagahara et al, 1995(Nagahara et al, , 1999. The intracellular localization of Str1 and Str2 of Arabidopsis has been investigated previously (Hatzfeld and Saito, 2000;Nakamura et al, 2000;Papenbrock and Schmidt, 2000a); however the localization results obtained by fusion with the green fluorescent protein (GFP) were ambiguous.…”