Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames IA50011, U.S.A.
Center for Biorenewable Chemicals, Iowa State University, Ames IA50011, U.S.A.
Biochem J. 2018 Dec 10;475(23):3861-3873. doi: 10.1042/BCJ20180470.
In plants and bacteria that use a Type II fatty acid synthase, isozymes of acyl-acyl carrier protein (ACP) thioesterase (TE) hydrolyze the thioester bond of acyl-ACPs, terminating the process of fatty acid biosynthesis. These TEs are therefore critical in determining the fatty acid profiles produced by these organisms. Past characterizations of a limited number of plant-sourced acyl-ACP TEs have suggested a thiol-based, papain-like catalytic mechanism, involving a triad of Cys, His, and Asn residues. In the present study, the sequence alignment of 1019 plant and bacterial acyl-ACP TEs revealed that the previously proposed Cys catalytic residue is not universally conserved and therefore may not be a catalytic residue. Systematic mutagenesis of this residue to either Ser or Ala in three plant acyl-ACP TEs, CvFatB1 and CvFatB2 from and CnFatB2 from , resulted in enzymatically active variants, demonstrating that this Cys residue (Cys348 in CvFatB2) is not catalytic. In contrast, the multiple sequence alignment, together with the structure modeling of CvFatB2, suggests that the highly conserved Asp309 and Glu347, in addition to previously proposed Asn311 and His313, may be involved in catalysis. The substantial loss of catalytic competence associated with site-directed mutants at these positions confirmed the involvement of these residues in catalysis. By comparing the structures of acyl-ACP TE and the 4-hydroxybenzoyl-CoA TE, both of which fold in the same hotdog tertiary structure and catalyze the hydrolysis reaction of thioester bond, we have proposed a two-step catalytic mechanism for acyl-ACP TE that involves an enzyme-bound anhydride intermediate.
在使用 II 型脂肪酸合酶的植物和细菌中，酰基辅酶 A-酰基载体蛋白 (ACP) 硫酯酶 (TE) 的同工酶水解酰基-ACP 的硫酯键，从而终止脂肪酸生物合成过程。因此，这些 TE 对于确定这些生物体产生的脂肪酸谱至关重要。过去对有限数量的植物来源的酰基-ACP TE 的特性研究表明，存在一种基于硫醇的、类似于木瓜蛋白酶的催化机制，涉及 Cys、His 和 Asn 残基的三联体。在本研究中，对 1019 种植物和细菌酰基-ACP TE 的序列比对表明，先前提出的 Cys 催化残基并非普遍保守，因此可能不是催化残基。在三种植物酰基-ACP TE（来自 的 CvFatB1 和 CvFatB2 以及来自 的 CnFatB2）中将该残基突变为 Ser 或 Ala 的系统突变，产生了具有酶活性的变体，表明该 Cys 残基（CvFatB2 中的 Cys348）不是催化残基。相比之下，多重序列比对以及 CvFatB2 的结构建模表明，高度保守的 Asp309 和 Glu347，除了先前提出的 Asn311 和 His313 外，可能参与催化。这些位置的定点突变导致催化能力显著丧失，证实了这些残基在催化中的作用。通过比较酰基-ACP TE 和 4-羟基苯甲酰辅酶 A TE 的结构，这两种酶都折叠在相同的热狗三级结构中并催化硫酯键的水解反应，我们提出了酰基-ACP TE 的两步催化机制，其中涉及酶结合的酸酐中间体。
