Cloning, sequencing and heterologous expression of a cDNA encoding pigeon liver carnitine acetyltransferase

Johnson, Todd M.; Kocher, Hans P.; Anderson, R.C.; Nemecek, Georgina M.

doi:10.1042/bj3050439

Cited by 10 publications

(3 citation statements)

References 38 publications

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“…If no other processing occurred, molecular sizes of 68191 Da for the peroxisomal enzyme and 66446Da for the mitochondrial enzyme would be expected from the deduced amino acid sequences. The apparent molecular masses measured by SDS/PAGE (64 kDa) were smaller than those predicted from the sequences, a tendency which was also demonstrated for the pigeon liver enzyme [31]. These results revealed that initiation of translation for peroxisomal carnitine acetyltransferase was at the second Met residue (Metl9) (Fig.…”

Section: Peroxisomal and Mitochondrial Carnitine Acetyltransferasesmentioning

confidence: 63%

Peroxisomal and Mitochondrial Carnitine Acetyltransferases of the n‐Alkane‐Assimilating Yeast Candida tropicalis

Kawachi

Atomi

Ueda

et al. 1996

European Journal of Biochemistry

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A genomic DNA clone encoding carnitine acetyltransferases (EC 2.3.1.7), localized in two subcellular organelles, peroxisomes and mitochondria of an n-alkane-assimilating yeast Candida tropicalis, was isolated from the yeast AEMBL library using a carnitine acetyltransferase cDNA probe. Nucleotide sequence analysis disclosed that the open reading frame was 1881 bp, corresponding to 627 amino acids with a molecular mass of 70760 Da. Comparison of the predicted amino acid sequence of the C. tropicalis enzyme with that of Saccharonzyces cerevisiae mitochondrial matrix carnitine acetyltransferase revealed 46.3 % identity. It was noticeable that the C. tropicalis enzymes had amino acid sequences similar to both proposed mitochondrial and peroxisomal targeting signals. When the C. tropicalis gene was expressed in S. cerevisiae using its own 5'-upstream region, a 12-fold increase in activity was observed. Western blot analysis revealed the presence of two major proteins whose sizes corresponded to the peroxisomal and mitochondrial proteins detected in C. tropicalis. This suggested that peroxisomal and mitochondrial carnitine acetyltransferases were encoded by one gene, as suggested for the S. cerevisiae enzyme. Furthermore, we have separated and purified these enzymes from peroxisomes and mitochondria of C. tropicalis, and analyzed the amino-terminal amino acid sequences of each. The amino-terminal sequence of the mitochondrial enzyme suggested that a signal sequence had been cleaved during translocation into mitochondria. Concerning the peroxisomal enzyme, the evidence obtained indicated that in vivo the translation was initiated at the second methionine of the open reading frame.

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Section: Peroxisomal and Mitochondrial Carnitine Acetyltransferasesmentioning

confidence: 63%

Peroxisomal and Mitochondrial Carnitine Acetyltransferases of the n‐Alkane‐Assimilating Yeast Candida tropicalis

Kawachi

Atomi

Ueda

et al. 1996

European Journal of Biochemistry

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“…(Esser et al, 1993;L07736), rat CPT-I-like (Yamazaki et a]., 1995; D43623), human CPT-I1 (Finocchiaro et al, 1991;M58581), rat CPT-I1 (Woeltje et al, 1990;J05470), mouse CPT-I1 (Gelb, 1993;U01163), rat COT (Choi et al, 1995;U26033), bovine COT (this study), mouse CAT (Brunner et a]., 1997; X85983), pigeon CAT (Johnson et al, 1995;U08229), yeast CAT (Kispal G., Tomcsanyi, T., Sumegi, B., Bock, I., Gajdos, G., Dietmayer, K. and Sandor, A., 1992;214021), human ChAT (Oda et al, 1992;S56138), rat ChAT (Ishii et al, 1990;P32738), pig ChAT (Berrard et a]., 1989; M27736), C. elegans ChAT (Alfonso et al, 1994;L08969) and D. melanogaster ChAT (Sugihara et a]., 1990; M63724) were aligned using Clustal V (Higgins et a]., 1992) (all accession numbers are GenBank entries with the exception of rat ChAT, which is a SwissProt database entry).…”

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confidence: 99%

cDNA Cloning, Recombinant Expression, and Site‐Directed Mutagenesis of Bovine Liver Carnitine Octanoyltransferase

Cronin¹

1997

European Journal of Biochemistry

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The cDNA for bovine liver carnitine octanoyltransferase (COT) has been cloned by a combination of Agtll library screening and 3' rapid amplification of cDNA ends (3'-RACE). The cDNA comprises 338 bases of 5' non-coding sequence, a reading frame of 1839 bases including the stop codon, and 820 bases of 3' non-coding DNA. The deduced amino acid sequence of 612 residues predicts a protein with a calculated mass of 70263 Da and PI 6.28. The enzyme was expressed in recombinant soluble form in Escherichia coli and was purified by a two-step procedure to near-homogeneity with a yield of purified protein of 2-3 mg/l culture. Recombinant COT had similar kinetic properties to those of the enzyme isolated directly from beef liver. Arg505 in COT, conserved in all reported camitine acyltransferase sequences but replaced by asparagine or isoleucine in the choline acetyltransferases, was converted to asparagine by site-directed mutagenesis. This single mutation resulted in a greater than 1650-fold increase in the K , value for COT towards carnitine, but had little effect on the value of k,,, or the K , value for the acyl-CoA substrate. In addition, although choline was an extremely poor substrate for COT, the k,J K , ratio towards this substrate was increased fourfold as a result of the mutation. These data support the notion that Arg505 in COT, and other carnitine acyltransferases, contributes to substrate binding by forming a salt bridge with the carboxylate moiety of carnitine.

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“…1) showed ∼ 53% nucleotide sequence identity to carnitine acetyltransferase genes from human (Corti et al 1994) and pigeon (Johnson et al 1995). Similar levels of identity were found across about two thirds of the sequence to choline acetyltransferase genes from pig (Berrard et al 1987(Berrard et al , 1989 and human (Lorenzi et al 1992).…”

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confidence: 49%

Anopheles stephensi Dox-A2 Shares Common Ancestry with Genes from Distant Groups of Eukaryotes Encoding a 26S Proteasome Subunit and Is in a Conserved Gene Cluster

Garvey

Malcolm

2000

J Mol Evol

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The sequence of a cloned Anopheles stephensi gene showed 72% inferred amino acid identity with Drosophila melanogaster Dox-A2 and 93% with its putative ortholog in Anopheles gambiae. Dox-A2 is the reported but herein disputed structural locus for diphenol oxidase A2. Database searches identified Dox-A2 related gene sequences from 15 non-insect species from diverse groups. Phylogenetic trees based on alignments of inferred protein sequences, DNA, and protein motif searches and protein secondary structure predictions produced results consistent with expectations for genes that are orthologous. The only inconsistency was that the C-terminus appears to be more primitive in the yeasts than in plants. In mammals, plants, and yeast these genes have been shown to code for a non-ATPase subunit of the PA700 (19S) regulatory complex of 26S proteasome. The analyses indicated that the insect genes contain no divergent structural features, which taken within an appraisal of all available data, makes the reported alternative function highly improbable. A plausible additional role, in which the 26S proteasome is implicated in regulation of phenol oxidase, would also apply to at least the mammalian genes. No function has yet been reported for the other included sequences. These were from genome projects and included Caenorhabiditus elegans, Arabidopsis thaliana, Fugu rubripes, and Toxoplasma gondii. A consensus of the results predicts a protein containing exceptionally long stretches of helix with a hydrophilic C-terminus. Phosphorylation site motifs were identified at two conserved positions. Possible SRY and GATA-1 binding motifs were found at conserved positions upstream of the mosquito genes. The location of A. stephensi Dox-A2 was determined by in situ hybridization at 34D on chromosome arm 3R. It is in a conserved gene cluster with respect to the other insects. However, the A. stephensi cluster contains a gene showing significant sequence identity to human and pigeon carnitine acetyltransferase genes, therefore showing divergence with the distal end of the D. melanogaster cluster.

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Cloning, sequencing and heterologous expression of a cDNA encoding pigeon liver carnitine acetyltransferase

Cited by 10 publications

References 38 publications

Peroxisomal and Mitochondrial Carnitine Acetyltransferases of the n‐Alkane‐Assimilating Yeast Candida tropicalis

Peroxisomal and Mitochondrial Carnitine Acetyltransferases of the n‐Alkane‐Assimilating Yeast Candida tropicalis

cDNA Cloning, Recombinant Expression, and Site‐Directed Mutagenesis of Bovine Liver Carnitine Octanoyltransferase

Anopheles stephensi Dox-A2 Shares Common Ancestry with Genes from Distant Groups of Eukaryotes Encoding a 26S Proteasome Subunit and Is in a Conserved Gene Cluster

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