PDF(Pubmed)
Abstract：
Acetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded β-barrel of the partially resolved BCCP domain. Disruption of β-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTβ-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTβ to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the \"swing domain model\" where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.
摘要：
乙酰辅酶A羧化酶（ACCs）将乙酰辅酶A转化为丙二酰辅酶A，脂肪酸生物合成和自养碳固定途径的关键步骤。三个功能不同的组件，生物素羧化酶（BC），生物素羧基载体蛋白(BCCP),和羧化酶（CT），以不同的组合分离或部分融合，形成异聚ACC。然而,合并BC-BCCP和单独CT的ACC尚未被识别，其催化机理尚不清楚。这里,我们从金氯氟菌中鉴定出两种BC亚型（BC1和BC2），一种丝状的缺氧光生体，采用3-羟基丙酸酯（3-HP）双循环而不是卡尔文循环进行自养碳固定。我们发现BC1具有融合的BC和BCCP结构域，其中BCCP可以在Lys553残基上被大肠杆菌或C.aurantiacusBirA生物素化。BC1和BC2的晶体结构在3.2和3.0分辨率下，分别,进一步揭示了两个BC1-BC同源二聚体的四聚体，和一个BC2同源二聚体，都表现出相似的BC架构。两个BC1-BC同源二聚体通过部分解析的BCCP结构域的八链β-桶连接。β-桶的破坏导致四聚体在溶液中解离成二聚体并降低生物素羧化酶活性。BCCP结构域的生物素化进一步促进BC1和CTβ-CTα相互作用，形成具有酶活性的ACC，其在体外将乙酰辅酶A转化为丙二酰辅酶A,并通过在大肠杆菌细胞中与重组丙二酰辅酶A还原酶共表达产生3-HP。这项研究揭示了一种异聚ACC，该ACC进化出融合的BC-BCCP，但分离了CTα和CTβ以完成ACC活性。IMPORTANCEAC-CoA羧化酶（ACC）催化脂肪酸生物合成中的限速步骤和各种生物体的自养碳固定途径，使它们成为针对各种感染和疾病的药物发现的有吸引力的目标。虽然对同聚ACC的结构研究，由具有三个亚基的单一蛋白质组成，揭示了“摆动域模型”，其中生物素羧基载体蛋白（BCCP）域在生物素羧化酶（BC）和羧化酶（CT）活性位点之间易位以促进反应，我们对异聚ACCs的亚基组成和催化机理的理解仍然有限.这里,我们从一种古老的缺氧光合细菌中鉴定出一种新的ACC，它进化出融合的BC和BCCP结构域，而是分离CT成分以形成具有酶活性的ACC，其在体外将乙酰辅酶A转化为丙二酰辅酶A,并通过在大肠杆菌细胞中与重组丙二酰辅酶A还原酶共表达产生3-羟基丙酸酯(3-HP)。这些发现扩展了异聚ACCs的多样性和分子进化，并为3-HP生物合成的潜在应用提供了结构基础。
