Biochemical and structural characterization of the arabidopsis bifunctional enzyme dethiobiotin synthetase-diaminopelargonic acid aminotransferase: Evidence for substrate channeling in biotin synthesis

D. Cobessi, C. Meinguet, J.-L. Ferrer, R. Dumas, V. Pautre, C. Alban

Research output: Contribution to journalArticle

Abstract

Diaminopelargonic acid aminotransferase (DAPA-AT) and dethiobiotin synthetase (DTBS) catalyze the antepenultimate and the penultimate steps, respectively, of biotin synthesis. Whereas DAPA-AT and DTBS are encoded by distinct genes in bacteria, in biotin-synthesizing eukaryotes (plants and most fungi), both activities are carried out by a single enzyme encoded by a bifunctional gene originating from the fusion of prokaryotic monofunctional ancestor genes. In few angiosperms, including Arabidopsis thaliana, this chimeric gene (named BIO3-BIO1) also produces a bicistronic transcript potentially encoding separate monofunctional proteins that can be produced following an alternative splicing mechanism. The functional significance of the occurrence of a bifunctional enzyme in biotin synthesis pathway in eukaryotes and the relative implication of each of the potential enzyme forms (bifunctional versus monofunctional) in the plant biotin pathway are unknown. In this study, we demonstrate that the BIO3-BIO1 fusion protein is the sole protein form produced by the BIO3-BIO1 locus in Arabidopsis. The enzyme catalyzes both DAPA-AT and DTBS reactions in vitro and is targeted to mitochondria in vivo. Our biochemical and kinetic characterizations of the pure recombinant enzyme show that in the course of the reaction, the DAPA intermediate is directly transferred from the DAPA-AT active site to the DTBS active site. Analysis of several structures of the enzyme crystallized in complex with and without its ligands reveals key structural elements involved for acquisition of bifunctionality and brings, together with mutagenesis experiments, additional evidences for substrate channeling.
Original languageEnglish
Pages (from-to)1608-1625
Number of pages18
JournalPlant Cell Physiol,
Volume24
Issue number4
DOIs
Publication statusPublished - 1 Apr 2012

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dethiobiotin synthetase
transaminases
biotin
Biotin
Transaminases
ligases
Arabidopsis
Acids
synthesis
acids
Enzymes
enzymes
Eukaryota
active sites
eukaryotic cells
Catalytic Domain
genes
Genes
Angiosperms
Proteins

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title = "Biochemical and structural characterization of the arabidopsis bifunctional enzyme dethiobiotin synthetase-diaminopelargonic acid aminotransferase: Evidence for substrate channeling in biotin synthesis",
abstract = "Diaminopelargonic acid aminotransferase (DAPA-AT) and dethiobiotin synthetase (DTBS) catalyze the antepenultimate and the penultimate steps, respectively, of biotin synthesis. Whereas DAPA-AT and DTBS are encoded by distinct genes in bacteria, in biotin-synthesizing eukaryotes (plants and most fungi), both activities are carried out by a single enzyme encoded by a bifunctional gene originating from the fusion of prokaryotic monofunctional ancestor genes. In few angiosperms, including Arabidopsis thaliana, this chimeric gene (named BIO3-BIO1) also produces a bicistronic transcript potentially encoding separate monofunctional proteins that can be produced following an alternative splicing mechanism. The functional significance of the occurrence of a bifunctional enzyme in biotin synthesis pathway in eukaryotes and the relative implication of each of the potential enzyme forms (bifunctional versus monofunctional) in the plant biotin pathway are unknown. In this study, we demonstrate that the BIO3-BIO1 fusion protein is the sole protein form produced by the BIO3-BIO1 locus in Arabidopsis. The enzyme catalyzes both DAPA-AT and DTBS reactions in vitro and is targeted to mitochondria in vivo. Our biochemical and kinetic characterizations of the pure recombinant enzyme show that in the course of the reaction, the DAPA intermediate is directly transferred from the DAPA-AT active site to the DTBS active site. Analysis of several structures of the enzyme crystallized in complex with and without its ligands reveals key structural elements involved for acquisition of bifunctionality and brings, together with mutagenesis experiments, additional evidences for substrate channeling.",
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Biochemical and structural characterization of the arabidopsis bifunctional enzyme dethiobiotin synthetase-diaminopelargonic acid aminotransferase : Evidence for substrate channeling in biotin synthesis. / Cobessi, D.; Meinguet, C.; Ferrer, J.-L.; Dumas, R.; Pautre, V.; Alban, C.

In: Plant Cell Physiol, , Vol. 24, No. 4, 01.04.2012, p. 1608-1625.

Research output: Contribution to journalArticle

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T1 - Biochemical and structural characterization of the arabidopsis bifunctional enzyme dethiobiotin synthetase-diaminopelargonic acid aminotransferase

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AU - Cobessi, D.

AU - Meinguet, C.

AU - Ferrer, J.-L.

AU - Dumas, R.

AU - Pautre, V.

AU - Alban, C.

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