Soly R R, Meighen E A
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
J Mol Biol. 1991 May 5;219(1):69-77. doi: 10.1016/0022-2836(91)90858-4.
Fatty acid activation, transfer, and reduction by the fatty acid reductase multienzyme complex from Photobacterium phosphoreum to generate fatty aldehydes for the luminescence reaction is regulated by the interaction of the synthetase and reductase subunits of this complex. Identification of the specific site involved in covalent transfer of the fatty acyl group between the sites of activation and reduction on the synthetase and reductase subunits, respectively, is a critical step in understanding how subunit interactions modulate the flow of fatty acyl groups through the fatty acid reductase complex. To accomplish this goal, the nucleotide sequence of the luxE gene coding for the acyl-protein synthetase subunit (373 amino acid residues) was determined and the conserved cysteinyl residues implicated in fatty acyl transfer identified. Using site-specific mutagenesis, each of the five conserved cysteine residues was converted to a serine residue, the mutated synthetases expressed in Escherichia coli, and the properties of the mutant proteins examined. On complementation of four of the mutants with the reductase subunit, the synthetase subunit was acylated and the acyl group could be reversibly transferred between the reductase and synthetase subunits, and fatty acid reductase activity was fully regenerated. As well, sensitivity of the acylated synthetases to hydroxylamine cleavage (under denaturation conditions to remove any conformational effects on reactivity) was retained, showing that a cysteine and not a serine residue was still acylated. However, substitution of a cysteine residue only ten amino acid residues from the carboxyl terminal (C364S) prevented acylation of the synthetase and regeneration of fatty acid reductase activity. Moreover, this mutant protein preserved its ability to activate fatty acid to fatty acyl-AMP but could not accept the acyl group from the reductase subunit, demonstrating that the C364S synthetase had retained its conformation and specifically lost the fatty acylation site. These results provide evidence that the flow of fatty acyl groups in the fatty acid reductase complex is modulated by interaction of the reductase subunit with a cysteine residue very close to the carboxyl terminal of the synthetase, which in turn acts as a flexible arm to transfer acyl groups between the sites of activation and reduction.
发光杆菌脂肪酸还原酶多酶复合体对脂肪酸进行激活、转移和还原以生成用于发光反应的脂肪醛，这一过程受该复合体合成酶和还原酶亚基相互作用的调控。确定分别参与合成酶和还原酶亚基上脂肪酸激活位点与还原位点之间脂肪酰基共价转移的特定位点，是理解亚基相互作用如何调节脂肪酰基通过脂肪酸还原酶复合体流动的关键一步。为实现这一目标，测定了编码酰基蛋白合成酶亚基（373个氨基酸残基）的luxE基因的核苷酸序列，并鉴定了与脂肪酰基转移有关的保守半胱氨酸残基。利用定点诱变技术，将五个保守半胱氨酸残基中的每一个都转化为丝氨酸残基，在大肠杆菌中表达突变的合成酶，并检测突变蛋白的性质。当四个突变体与还原酶亚基互补时，合成酶亚基被酰化，酰基可在还原酶和合成酶亚基之间可逆转移，脂肪酸还原酶活性完全恢复。此外，酰化合成酶对羟胺裂解（在变性条件下以消除对反应性的任何构象影响）的敏感性得以保留，表明仍然是半胱氨酸残基被酰化而不是丝氨酸残基。然而，将仅位于羧基末端十个氨基酸残基处的半胱氨酸残基进行替换（C364S）会阻止合成酶的酰化以及脂肪酸还原酶活性的恢复。此外，这种突变蛋白保留了将脂肪酸激活为脂肪酰 - AMP的能力，但不能从还原酶亚基接受酰基，这表明C364S合成酶保留了其构象，并且特异性地失去了脂肪酰化位点。这些结果证明，脂肪酸还原酶复合体中脂肪酰基的流动受还原酶亚基与合成酶羧基末端非常接近的半胱氨酸残基相互作用的调节，该半胱氨酸残基反过来充当一个灵活的臂，在激活位点和还原位点之间转移酰基。
