Tissot G, Pepin R, Job D, Douce R, Alban C
Laboratoire Mixte CNRS/Rhône-Poulenc (UMR41 associée au Centre National de la Recherche Scientifique), Rhône-Poulenc Agrochimie, Lyon, France.
Eur J Biochem. 1998 Dec 1;258(2):586-96. doi: 10.1046/j.1432-1327.1998.2580586.x.
Holocarboxylase synthetases (HCSs) are key enzymes in biotin utilisation in both prokaryotes and eukaryotes. In a previous study, we demonstrated that, in plants, HCS activity is localised in cytosol, chloroplasts and mitochondria. We also described the cloning and sequencing of a full-length cDNA encoding an Arabidopsis thaliana HCS isoform with a putative organelle-transit peptide. In the study reported here, this cDNA was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated using an efficient three-step purification procedure. Polyclonal antibodies raised against pure HCS were produced to elucidate the subcellular localisation of this protein. Immunodetection carried out by Western blotting of isolated pea leaf subcellular compartments specifically revealed a single polypeptide that was ascribed to the chloroplast compartment. Immunocytochemistry of thin-cut sections from tobacco leaves, transformed by the complete coding sequence of A. thaliana HCS cDNA via Agrobacterium tumefaciens, confirmed that the enzyme encoded by this cDNA is the chloroplastic isoform. Moreover, physicochemical, biochemical and kinetic properties of the pure recombinant HCS were determined. The native recombinant enzyme is a 37-kDa monomer. In contrast to the major part of HCS activity measured in leaf extracts, the recombinant chloroplastic enzyme did not require addition of Mg2+ to be fully active, but was substantially inhibited by EDTA. This suggested that the chloroplastic HCS may contain a tightly-bound divalent cation required for enzyme activity. The recombinant enzyme was able to biotinylate efficiently apo-biotin carboxyl carrier protein (BCCP) from E. coli and apo-methylcrotonoyl-CoA carboxylase (MCCase) from A. thaliana. Apparent Km values for the enzyme substrates D-biotin, ATP and apo-MCCase were found to be 130 nM, 4.4 microM and 32 microM, respectively. Steady-state kinetic analyses of the HCS-catalysed reaction were investigated with respect to reaction mechanism and inhibition by AMP, one of the end-products of the enzyme-catalysed reaction. Substrate interaction and product inhibition patterns indicated that ATP and D-biotin bind sequentially, in an ordered manner, to the enzyme and that ATP or D-biotin and apo-BCCP bind in ping-pong fashion.
全羧化酶合成酶（HCSs）是原核生物和真核生物中生物素利用的关键酶。在之前的一项研究中，我们证明，在植物中，HCS活性定位于细胞质、叶绿体和线粒体中。我们还描述了编码拟南芥HCS同工型且带有假定细胞器转运肽的全长cDNA的克隆和测序。在本文报道的研究中，该cDNA被用于构建一个高产大肠杆菌菌株。使用高效的三步纯化程序分离重组酶。制备了针对纯HCS的多克隆抗体，以阐明该蛋白的亚细胞定位。通过对分离的豌豆叶亚细胞区室进行蛋白质印迹免疫检测，特异性地揭示了一条单一的多肽，它被归属于叶绿体区室。对经根癌农杆菌介导转入拟南芥HCS cDNA完整编码序列的烟草叶片薄切片进行免疫细胞化学分析，证实该cDNA编码的酶是叶绿体同工型。此外，还测定了纯重组HCS的物理化学、生化和动力学性质。天然重组酶是一种37 kDa的单体。与在叶片提取物中测得的大部分HCS活性不同，重组叶绿体酶不需要添加Mg2+就能完全激活，但会被EDTA显著抑制。这表明叶绿体HCS可能含有酶活性所需的紧密结合的二价阳离子。重组酶能够有效地将大肠杆菌的脱辅基生物素羧基载体蛋白（BCCP）和拟南芥的脱辅基甲基巴豆酰辅酶A羧化酶（MCCase）生物素化。发现该酶底物D-生物素、ATP和脱辅基MCCase的表观Km值分别为130 nM、4.4 μM和32 μM。针对酶促反应机制以及该酶促反应终产物之一AMP的抑制作用，对HCS催化反应进行了稳态动力学分析。底物相互作用和产物抑制模式表明，ATP和D-生物素以有序的方式依次结合到酶上，并且ATP或D-生物素与脱辅基BCCP以乒乓模式结合。
