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.

Arabidopsis RIBA Proteins: Two out of Three Isoforms Have Lost Their Bifunctional Activity in Riboflavin Biosynthesis

“…Thus, sequestration and protection of labile metabolic intermediates can improve metabolic channeling as demonstrated recently for bifunctional BIO3-BIO1 in Arabidopsis biotin synthesis [27]. …”

Plant amino acid-derived vitamins: biosynthesis and function

“…SAM is also involved in two steps of biotin biosynthesis. While it first acts as an amino group donor for the diaminopelargonic acid aminotransferase (Cobessi et al 2012), it also generates an adenosyl radical and acts as a sulfur donor closing the thiophene ring of biotin by the insertion of a sulfur atom in the last step of the pathway (biotin synthase) (Patton et al 1998;Roje 2006). Therefore, either incorporated to its backbone or as donors of amino, sulfur or methyl groups, amino acids play crucial roles in vitamin biosynthesis.…”

Expanding functional repertoires of fungal peroxisomes: contribution to growth and survival processes

“…The BIO3 and BIO1 genes are unidirectionally aligned and expressed as a chimeric transcript, resulting in the production of Bio3-Bio1 as a bifunctional protein catalyzing desthiobiotin (DTB) synthase and 7, 8-diaminopelargonic acid (DAPA) synthase reactions (Muralla et al, 2008). Bio3-Bio1 contains a mitochondrial targeting sequence (MTS) and localizes in mitochondria (Muralla et al, 2008; Cobessi et al, 2012). The Bio2 protein, a biotin synthase catalyzing the conversion of DTB to biotin, also contains a MTS and must be mitochondrially localized for biotin prototrophy (Baldet et al, 1997; Picciocchi et al, 2001; Arnal et al, 2006).…”