Dictyostelium acetoacetyl-CoA thiolase is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol

Isezaki, Nana; Sekiba, Atsushi; Itagaki, Shoko; Nagayama, Koki; Ochiai, Hiroshi; Ohmachi, Tetsuo

doi:10.1099/mic.0.000102

“…Currently, there are no data (such as cDNAs) pointing to D. discoideum dUTPase isoforms, but these cannot be ruled out as there are examples in D. discoideum where two versions of a protein are generated from single copy genes. These include the peroxisomal and mitochondrial isoforms of the enzyme acetoacetyl-CoA thiolase (DDB_G0271544) that arise from alternative start codons [25], and (cytoplasmic and mitochondrial) isoforms of fumarase (DDB_G0280495), inferred from expressed sequence tag data. Fumarase isoforms have been documented extensively in yeast and mammals (reviewed in 26).…”

Section: Limitationsmentioning

confidence: 99%

Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

Chia

¹

,

Inoguchi

²

,

Varon

³

et al. 2020

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Objective: The nuclear and mitochondrial genomes of Dictyostelium discoideum, a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectively. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. Results:The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the shortened N-termini that arose from the loss of residues 1-37. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

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“…CoA thiolase (DDB_G0271544) that arise from alternative start codons [25] and (cytoplasmic and mitochondrial) isoforms of fumarase (DDB_G0280495), inferred from expressed sequence tag data. residues.…”

Section: A Single Gene Codes For An Active Enzyme In the Homotrimericmentioning

confidence: 99%

Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

Chia

¹

,

Inoguchi

²

,

Varon

³

et al. 2019

Preprint

0

View full text Add to dashboard Cite

Objective The nuclear and mitochondrial genomes of Dictyostelium discoideum , a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectivey. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. Results The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the truncated N-termini. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

show abstract

“…These include the peroxisomal and mitochondrial isoforms of the enzyme acetoacetyl-CoA thiolase (DDB_G0271544) that arise from alternative start codons (25), and (cytoplasmic and mitochondrial) isoforms of fumarase (DDB_G0280495), inferred from expressed sequence tag data. Fumarase isoforms have been documented extensively in yeast and mammals (reviewed in 26).…”

Section: Limitationsmentioning

confidence: 99%

Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

Chia

¹

,

Inoguchi

²

,

Varon

³

et al. 2019

Preprint

0

View full text Add to dashboard Cite

Objective The nuclear and mitochondrial genomes of Dictyostelium discoideum , a unicellular eukaryote, have relatively high A+T-contents of 77.5% and 72.65%, respectivey. To begin to investigate how the pyrimidine biosynthetic pathway fulfills the demand for dTTP, we determined the catalytic properties and structure of the key enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) that hydrolyzes dUTP to dUMP, the precursor of dTTP. Results The annotated genome of D. discoideum identifies a gene encoding a polypeptide containing the five conserved motifs of homotrimeric dUTPases. Recombinant proteins, comprised of either full-length or core polypeptides with all conserved motifs but lacking residues 1-37 of the N-terminus, were active dUTPases. Crystallographic analyses of the core enzyme indicated that the C-termini, normally flexible, were constrained by interactions with the truncated N-termini. This allowed greater access of dUTP to active sites, resulting in enhanced catalytic parameters. A tagged protein comprised of the N-terminal forty amino acids of dUTPase fused to green fluorescent protein (GFP) was expressed in D. discoideum cells. Supporting a prediction of mitochondrial targeting information within the N-terminus, localization and subcellular fractionation studies showed GFP to be in mitochondria. N-terminal sequencing of immunoprecipitated GFP revealed the loss of the dUTPase sequence upon import into the organelle.

show abstract

Dictyostelium acetoacetyl-CoA thiolase is a dual-localizing enzyme that localizes to peroxisomes, mitochondria and the cytosol

Cited by 3 publications

References 39 publications

Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

Mitochondrial localization of Dictyostelium discoideum dUTPase mediated by its N-terminus

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