Plant mercaptopyruvate sulfurtransferases: molecular cloning, subcellular localization and enzymatic activities.
Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) and thiosulfate sulfurtransferase (TST, rhodanese, EC 2.8.1.1) are evolutionarily related enzymes that catalyze the transfer of sulfur ions from mercaptopyruvate and thiosulfate, respectively, to cyanide ions. We have isolated and characterized two cDNAs, AtMST1 and AtMST2, that are Arabidopsis homologs of TST and MST from other organisms. Deduced amino-acid sequences showed similarity to each other, although MST1 has a N-terminal extension of 57 amino acids containing a targeting sequence. MST1 and MST2 are located in mitochondria and cytoplasm, respectively, as shown by immunoblot analysis of subcellular fractions and by green fluorescent protein (GFP) analysis. However, some regions of MST1 fused to GFP were found to target not only mitochondria, but also chloroplasts, suggesting that the regions on the targeting sequence recognized by protein import systems of mitochondria and chloroplasts are not identical. Recombinant proteins, expressed in Escherichia coli, exhibited MST/TST activity ratios determined from kcat/Km values of 11 and 26 for MST1 and MST2, respectively. This indicates that the proteins encoded by both AtMST1 and AtMST2 are MST rather than TST type. One of the hypotheses proposed so far for the physiological function of MST and TST concerns iron-sulfur cluster assembly. In order to address this possibility, a T-DNA insertion Arabidopsis mutant, in which the AtMST1 was disrupted, was isolated by PCR screening of T-DNA mutant libraries. However, the mutation had no effect on levels of iron-sulfur enzyme activities, suggesting that MST1 is not directly involved in iron-sulfur cluster assembly.
Nakamura
T
T
Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
Yamaguchi
Y
Y
Sano
H
H
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology*; D013466:Sulfurtransferases / Q000235:genetics* / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D007506:Iron-Sulfur Proteins; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; C021550:3-mercaptopyruvate sulphurtransferase