Literature summary for 3.1.3.78 extracted from

Aleman, A.; Rodriguez-Escudero, I.; Mallo, G.V.; Cid, V.J.; Molina, M.; Rotger, R.
The amino-terminal non-catalytic region of Salmonella typhimurium SigD affects actin organization in yeast and mammalian cells (2005), Cell. Microbiol., 7, 1432-1446.

Cloned(Commentary)

Cloned (Comment)	Organism
(PCR) amplification from the SigD open reading frame from S. enterica ser. typhimurium C53 genomic DNA. 2 types plasmids: first type based on the pEG-KG vector to express GST fusion proteins in yeast, second type based on pGEX-KG for expression of GST-fused proteins in bacteria. transformation of Escherichia coli DH5alpha cells (used for molceularbiological methods and expression). Transformation of Saccharomyces cerevisiae YPH499 (used for SigD functional analyzes as a model organism). transfection of human epithelial cell line HeLa (ATCC CCL2). yeast: endogenous SigD expression results in severe growth inhibition, allele mutant in catalytic site or deletion of whole C-terminal phosphatase domain inhibit growth by loss of actin polarization and precluding the budding process. HeLa: expression of same sigD alleles causes loss of ability of depleting phosphatidylinositol 4,5-bisphosphate from the plasma membrane, actin fibres still disappear and lose their adherence. region of 25 amino acids localized (residues 118142) essentially required for the effect of SigD on actin in HeLa cells. SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity	Salmonella enterica subsp. enterica serovar Typhimurium

Cloned (Comment)

Organism

(PCR) amplification from the SigD open reading frame from S. enterica ser. typhimurium C53 genomic DNA. 2 types plasmids: first type based on the pEG-KG vector to express GST fusion proteins in yeast, second type based on pGEX-KG for expression of GST-fused proteins in bacteria. transformation of Escherichia coli DH5alpha cells (used for molceularbiological methods and expression). Transformation of Saccharomyces cerevisiae YPH499 (used for SigD functional analyzes as a model organism). transfection of human epithelial cell line HeLa (ATCC CCL2). yeast: endogenous SigD expression results in severe growth inhibition, allele mutant in catalytic site or deletion of whole C-terminal phosphatase domain inhibit growth by loss of actin polarization and precluding the budding process. HeLa: expression of same sigD alleles causes loss of ability of depleting phosphatidylinositol 4,5-bisphosphate from the plasma membrane, actin fibres still disappear and lose their adherence. region of 25 amino acids localized (residues 118142) essentially required for the effect of SigD on actin in HeLa cells. SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity

Salmonella enterica subsp. enterica serovar Typhimurium

Protein Variants

Protein Variants	Comment	Organism
C462A	recombinant protein	Salmonella enterica subsp. enterica serovar Typhimurium
DELTA1-135-DELTA118-142	recombinant protein, Saccharomyces cerevisiae: no effect in Saccharomyces cerevisiae, HeLa: no effect (Green Fluorescent Protein linked) in HeLa	Salmonella enterica subsp. enterica serovar Typhimurium
DELTA1-165	recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, HeLa (Green Fluorescent Protein linked): loss of adherence and strong impact on actin filaments	Salmonella enterica subsp. enterica serovar Typhimurium
DELTA1-351	recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, HeLa (Green Fluorescent Protein linked): loss of adherence and strong impact on actin filaments	Salmonella enterica subsp. enterica serovar Typhimurium
DELTA118-142	recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, residues 118142 crucial for toxicity of SigD in the yeast cells, affecting both phosphatase activity and actin depolarization events, no effect (Green Fluorescent Protein linked) in HeLa	Salmonella enterica subsp. enterica serovar Typhimurium
DELTA352-563	recombinant protein, no effect in Saccharomyces cerevisiae, no effect in HeLa (Green Fluorescent Protein linked)	Salmonella enterica subsp. enterica serovar Typhimurium
R468A	recombinant protein, Saccharomyces cerevisiae: strong impact on actin filaments and budding, HeLa (Green Fluorescent Protein linked): loss of adherence and strong impact on actin filaments	Salmonella enterica subsp. enterica serovar Typhimurium

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
phosphatidylinositol 4,5-bisphosphate + H2O	Salmonella enterica subsp. enterica serovar Typhimurium	-	phosphatidylinositol 5-monophosphate + phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Salmonella enterica subsp. enterica serovar Typhimurium	-	-	-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.0000003	-	approx., C462A mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)	Salmonella enterica subsp. enterica serovar Typhimurium
0.0000005	-	approx., R468A mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)	Salmonella enterica subsp. enterica serovar Typhimurium
0.0000007	-	approx., GST, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)	Salmonella enterica subsp. enterica serovar Typhimurium
0.0000011	-	approx., DELTA1-351 mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)	Salmonella enterica subsp. enterica serovar Typhimurium
0.0000015	-	approx., DELTA352-563 mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)	Salmonella enterica subsp. enterica serovar Typhimurium
0.0000032	-	approx., DELTA118-142 mutant, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)	Salmonella enterica subsp. enterica serovar Typhimurium
0.0000063	-	approx., SigD, expressed in Escherichia coli DH5alpha, GST-tagged, malachite-green phosphatase assay (phosphatidylinositol 3,4,5-trisphosphate)	Salmonella enterica subsp. enterica serovar Typhimurium

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
phosphatidylinositol 3,4,5-trisphosphate + H2O	-	Salmonella enterica subsp. enterica serovar Typhimurium	phosphatidylinositol 3,5-bisphosphate + phosphate	-	?
phosphatidylinositol 4,5-bisphosphate + H2O	-	Salmonella enterica subsp. enterica serovar Typhimurium	phosphatidylinositol 5-monophosphate + phosphate	-	?

Synonyms

Synonyms	Comment	Organism
phosphoinositide phosphatase	-	Salmonella enterica subsp. enterica serovar Typhimurium
SigD	-	Salmonella enterica subsp. enterica serovar Typhimurium