Fetal mouse selenophosphate synthetase 2 (SPS2): Characterization of the cysteine mutant form overproduced in a baculovirus-insect cell system

Ick Young Kim, M. Jorge Guimarães, Albert Zlotnik, J. Fernando Bazan, Thressa C. Stadtman

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

A novel gene detected in mouse embryonic sites of hematopoiesis was cloned and shown to be a eukaryotic analog of the Escherichia coli selenophosphate synthetase gene. Unlike the E. coli enzyme, which is not a selenoprotein, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the gene in a position corresponding to the essential cysteine of the E. coli enzyme. An ionized selenol group in place of a cysteine sulfhydryl group could render this mammalian selenocysteine-containing enzyme a more active catalyst. The native cDNA clone and also a mutant form containing a TGC (cysteine) codon in place of TGA were expressed in a baculovirus-insect cell system. Based on recovery of purified proteins, expression of the mutant enzyme was about 40 times higher than wild-type enzyme. The cysteine mutant enzyme exhibited selenophosphate synthetase activity in the assay that measures selenide- dependent AMP formation from ATP. Although expression of wild-type enzyme has not been optimized, the mutant form of the fetal mouse enzyme can be produced in amounts sufficient for isolation in homogeneous form and precise physicochemical and mechanistic studies allowing direct comparison with the analogous cysteine-containing prokaryotic enzyme.

Original languageEnglish
Pages (from-to)418-421
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume94
Issue number2
DOIs
Publication statusPublished - 1997 Jan 21
Externally publishedYes

Fingerprint

Baculoviridae
Cysteine
Insects
Enzymes
Selenocysteine
Escherichia coli
Mouse Sephs2 protein
Selenoproteins
Genes
Terminator Codon
Hematopoiesis
Mutant Proteins
Adenosine Monophosphate
Codon
Open Reading Frames
Complementary DNA
Clone Cells
Adenosine Triphosphate

Keywords

  • eukaryotic selenophosphate synthetase

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Fetal mouse selenophosphate synthetase 2 (SPS2) : Characterization of the cysteine mutant form overproduced in a baculovirus-insect cell system. / Kim, Ick Young; Guimarães, M. Jorge; Zlotnik, Albert; Bazan, J. Fernando; Stadtman, Thressa C.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 94, No. 2, 21.01.1997, p. 418-421.

Research output: Contribution to journalArticle

@article{7ac45e653d8c40529eb7bf37c309a1db,
title = "Fetal mouse selenophosphate synthetase 2 (SPS2): Characterization of the cysteine mutant form overproduced in a baculovirus-insect cell system",
abstract = "A novel gene detected in mouse embryonic sites of hematopoiesis was cloned and shown to be a eukaryotic analog of the Escherichia coli selenophosphate synthetase gene. Unlike the E. coli enzyme, which is not a selenoprotein, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the gene in a position corresponding to the essential cysteine of the E. coli enzyme. An ionized selenol group in place of a cysteine sulfhydryl group could render this mammalian selenocysteine-containing enzyme a more active catalyst. The native cDNA clone and also a mutant form containing a TGC (cysteine) codon in place of TGA were expressed in a baculovirus-insect cell system. Based on recovery of purified proteins, expression of the mutant enzyme was about 40 times higher than wild-type enzyme. The cysteine mutant enzyme exhibited selenophosphate synthetase activity in the assay that measures selenide- dependent AMP formation from ATP. Although expression of wild-type enzyme has not been optimized, the mutant form of the fetal mouse enzyme can be produced in amounts sufficient for isolation in homogeneous form and precise physicochemical and mechanistic studies allowing direct comparison with the analogous cysteine-containing prokaryotic enzyme.",
keywords = "eukaryotic selenophosphate synthetase",
author = "Kim, {Ick Young} and Guimar{\~a}es, {M. Jorge} and Albert Zlotnik and Bazan, {J. Fernando} and Stadtman, {Thressa C.}",
year = "1997",
month = "1",
day = "21",
doi = "10.1073/pnas.94.2.418",
language = "English",
volume = "94",
pages = "418--421",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "2",

}

TY - JOUR

T1 - Fetal mouse selenophosphate synthetase 2 (SPS2)

T2 - Characterization of the cysteine mutant form overproduced in a baculovirus-insect cell system

AU - Kim, Ick Young

AU - Guimarães, M. Jorge

AU - Zlotnik, Albert

AU - Bazan, J. Fernando

AU - Stadtman, Thressa C.

PY - 1997/1/21

Y1 - 1997/1/21

N2 - A novel gene detected in mouse embryonic sites of hematopoiesis was cloned and shown to be a eukaryotic analog of the Escherichia coli selenophosphate synthetase gene. Unlike the E. coli enzyme, which is not a selenoprotein, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the gene in a position corresponding to the essential cysteine of the E. coli enzyme. An ionized selenol group in place of a cysteine sulfhydryl group could render this mammalian selenocysteine-containing enzyme a more active catalyst. The native cDNA clone and also a mutant form containing a TGC (cysteine) codon in place of TGA were expressed in a baculovirus-insect cell system. Based on recovery of purified proteins, expression of the mutant enzyme was about 40 times higher than wild-type enzyme. The cysteine mutant enzyme exhibited selenophosphate synthetase activity in the assay that measures selenide- dependent AMP formation from ATP. Although expression of wild-type enzyme has not been optimized, the mutant form of the fetal mouse enzyme can be produced in amounts sufficient for isolation in homogeneous form and precise physicochemical and mechanistic studies allowing direct comparison with the analogous cysteine-containing prokaryotic enzyme.

AB - A novel gene detected in mouse embryonic sites of hematopoiesis was cloned and shown to be a eukaryotic analog of the Escherichia coli selenophosphate synthetase gene. Unlike the E. coli enzyme, which is not a selenoprotein, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the gene in a position corresponding to the essential cysteine of the E. coli enzyme. An ionized selenol group in place of a cysteine sulfhydryl group could render this mammalian selenocysteine-containing enzyme a more active catalyst. The native cDNA clone and also a mutant form containing a TGC (cysteine) codon in place of TGA were expressed in a baculovirus-insect cell system. Based on recovery of purified proteins, expression of the mutant enzyme was about 40 times higher than wild-type enzyme. The cysteine mutant enzyme exhibited selenophosphate synthetase activity in the assay that measures selenide- dependent AMP formation from ATP. Although expression of wild-type enzyme has not been optimized, the mutant form of the fetal mouse enzyme can be produced in amounts sufficient for isolation in homogeneous form and precise physicochemical and mechanistic studies allowing direct comparison with the analogous cysteine-containing prokaryotic enzyme.

KW - eukaryotic selenophosphate synthetase

UR - http://www.scopus.com/inward/record.url?scp=0000301355&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0000301355&partnerID=8YFLogxK

U2 - 10.1073/pnas.94.2.418

DO - 10.1073/pnas.94.2.418

M3 - Article

C2 - 9012797

AN - SCOPUS:0000301355

VL - 94

SP - 418

EP - 421

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 2

ER -