Biochemical and Structural Characterization of the Arabidopsis Bifunctional Enzyme Dethiobiotin Synthetase-Diaminopelargonic Acid Aminotransferase: Evidence for Substrate Channeling in Biotin Synthesis
Biochemical and Structural Characterization of the Arabidopsis Bifunctional Enzyme Dethiobiotin Synthetase-Diaminopelargonic Acid Aminotransferase: Evidence for Substrate Channeling in Biotin Synthesis
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DOI：
10.1105/tpc.112.097675
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发表时间：
2012-04-01
期刊：
影响因子：
11.6
通讯作者：
Alban, Claude
中科院分区：
文献类型：
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作者：
Cobessi, David;Dumas, Renaud;Alban, Claude
二氨基壬酸转氨酶 (DAPA-AT) 和脱硫生物素合成酶 (DTBS) 分别催化生物素合成的倒数第二步和倒数第二步。虽然 DAPA-AT 和 DTBS 在细菌中由不同的基因编码，但在生物素合成真核生物（植物和大多数真菌）中，这两种活性均由源自原核单功能祖先基因融合的双功能基因编码的单一酶进行。在包括拟南芥在内的少数被子植物中，这种嵌合基因（称为 BIO3-BIO1）也会产生双顺反子转录本，该转录本可能编码单独的单功能蛋白质，这些蛋白质可以通过替代剪接机制产生。真核生物生物素合成途径中双功能酶出现的功能意义以及植物生物素途径中每种潜在酶形式（双功能与单功能）的相对含义尚不清楚。在这项研究中，我们证明 BIO3-BIO1 融合蛋白是拟南芥中 BIO3-BIO1 基因座产生的唯一蛋白质形式。该酶在体外催化 DAPA-AT 和 DTBS 反应，并在体内靶向线粒体。我们对纯重组酶的生化和动力学表征表明，在反应过程中，DAPA 中间体直接从 DAPA-AT 活性位点转移到 DTBS 活性位点。对有或没有配体的复合物中结晶的酶的几种结构的分析揭示了获得双功能性所涉及的关键结构元件，并与诱变实验一起提供了底物通道的额外证据。
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.