Selenocysteine incorporation in Kinetoplastid: selenophosphate synthetase (SELD) from Leishmania major and Trypanosoma brucei.
Selenophosphate synthetase (EC 2.7.9.3), the product of the selD gene, produces the biologically active selenium donor compound, monoselenophosphate, from ATP and selenide, for the synthesis of selenocysteine. The kinetoplastid Leishmania major and Trypanosoma brucei selD genes were cloned and the SELD protein overexpressed and purified to apparent homogeneity. The selD gene in L. major and T. brucei are respectively 1197 and 1179 bp long encoding proteins of 399 and 393 amino acids with molecular masses of 42.7 and 43 kDa. The molecular mass of 100 kDa for both (L. major and T. brucei) SELDs is consistent with dimeric proteins. The kinetoplastid selD complement Escherichia coli (WL400) selD deletion confirming it is a functional enzyme and the specific activity of these enzymes was determined. A conserved Cys residue was identified both by multiple sequence alignment as well as by functional complementation and activity assay of the mutant (Cys to Ala) forms of the SELD identifying this residue as essential for the catalytic function.
Sculaccio
S A
SA
Physics Institute of São Carlos, University of São Paulo (USP), Av. Trabalhador Sãocarlense 400, 13566-590 São Carlos-SP, Brazil.
Rodrigues
E M
EM
Cordeiro
A T
AT
Magalhães
A
A
Braga
A L
AL
Alberto
E E
EE
Thiemann
O H
OH
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D005816:Genetic Complementation Test; D018320:Leishmania major / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017279:Selenocysteine / Q000378:metabolism*; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000378:metabolism
D015800:Protozoan Proteins; D017279:Selenocysteine; D010770:Phosphotransferases; C062762:selenophosphate synthetase; D003545:Cysteine