Sulphonation of dehydroepiandrosterone and neurosteroids: molecular cloning, expression, and functional characterization of a novel zebrafish SULT2 cytosolic sulphotransferase.
By searching the zebrafish EST (expressed-sequence tag) database, we have identified two partial cDNA clones encoding the 5' and 3' regions of a putative zebrafish sulphotransferase (ST). Using the reverse transcription-PCR technique, a full-length cDNA encoding this zebrafish ST was successfully cloned. Sequence analysis revealed that this novel zebrafish ST displays 44%, 43% and 40% amino acid identity with mouse SULT2B1, human SULT2B1b and human SULT2A1 ST respectively. This zebrafish ST therefore appears to belong to the SULT2 cytosolic ST gene family. Recombinant zebrafish ST, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as a 34 kDa protein upon SDS/PAGE. Purified zebrafish ST displayed a strong sulphonating activity toward DHEA (dehydroepiandrosterone), with a optimum pH of 9.5. The enzyme also exhibited activities toward several neurosteroids with differential K(m) and V(max) values. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 degrees C and 43 degrees C. Among ten different divalent metal cations tested, Fe2+ and Cd(2+ exhibited small, but significant, stimulatory effects, whereas Hg2+ and Cu2+ displayed considerably stronger inhibitory effects on the DHEA-sulphonating activity of the enzyme. These results constitute the first study on the molecular cloning, expression, and characterization of a zebrafish cytosolic SULT2 ST.
Sugahara
Takuya
T
Biomedical Research Center, The University of Texas Health Center, Tyler, TX 75708, USA.
Yang
Yuh-Shyong
YS
Liu
Chau-Ching
CC
Pai
T Govind
TG
Liu
Ming-Cheh
MC
D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D003687:Dehydroepiandrosterone / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013256:Steroids / Q000378:metabolism*; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000378:metabolism; D015238:Sulfotransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D029961:Zebrafish Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D002413:Cations, Divalent; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013256:Steroids; D013451:Sulfonic Acids; D029961:Zebrafish Proteins; D003687:Dehydroepiandrosterone; D015238:Sulfotransferases; C484038:sult2st1 protein, zebrafish