Mutagenesis of rat acyl-CoA synthetase 4 indicates amino acids that contribute to fatty acid binding.
Mutagenesis of rat acyl-CoA synthetase 4 indicates amino acids that contribute to fatty acid binding.
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大鼠酰基辅酶 A 合成酶 4 的诱变表明有助于脂肪酸结合的氨基酸。
DOI：
10.1016/j.bbalip.2006.09.016
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发表时间：
2007
期刊：
影响因子：
--
通讯作者：
Coleman,RosalindA
中科院分区：
文献类型：
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作者：
Stinnett,Lori;Lewin,TalM;Coleman,RosalindA
虽然五种哺乳动物长链酰基辅酶A合成酶（ACSL）中的每一种都可以结合12至22个碳原子的饱和和不饱和脂肪酸，但ACSL 4更喜欢长链多不饱和脂肪酸。为了更好地了解ACSL 4脂肪酸结合，我们基于与从嗜热栖热菌HB 8结晶的ttLC-FACS相关的序列比对的诱变方法。选择用于诱变的四个残基对应于ttLC-FACS中包含脂肪酸结合口袋的残基;第五个残基与被认为参与大肠杆菌酰基辅酶A合成酶FadD的脂肪酸选择性的区域对齐。在假定的脂肪酸结合口袋G401 L的入口处改变氨基酸，导致失活的酶。突变口袋入口附近的残基，L399 M，没有显着改变酶活性，但突变的口袋，S291 Y，疏水末端的残基，改变ACSL 4的偏好为20：5和22：6，并增加其表观Km为ATP。突变一个先前被认为对脂肪酸结合重要的区域中的位点也改变了20：4和20：5的激活。这些研究表明，ACSL 4对长链多不饱和脂肪酸的偏好可以通过改变特定的氨基酸残基来改变。
Although each of the five mammalian long-chain acyl-CoA synthetases (ACSL) can bind saturated and unsaturated fatty acids ranging from 12 to 22 carbons, ACSL4 prefers longer chain polyunsaturated fatty acids. In order to gain a better understanding of ACSL4 fatty acid binding, we based a mutagenesis approach on sequence alignments related to ttLC-FACS crystallized from Thermus thermophilus HB8. Four residues selected for mutagenesis corresponded to residues in ttLC-FACS that comprise the fatty acid binding pocket; the fifth residue aligned with a region thought to be involved in fatty acid selectivity of the Escherichia coli acyl-CoA synthetase, FadD. Changing an amino acid at the entry of the putative fatty acid binding pocket, G401L, resulted in an inactive enzyme. Mutating a residue near the pocket entry, L399M, did not significantly alter enzyme activity, but mutating a residue at the hydrophobic terminus of the pocket, S291Y, altered ACSL4's preference for 20:5 and 22:6 and increased its apparent Kmfor ATP. Mutating a site in a region previously identified as important for fatty acid binding also altered activation of 20:4 and 20:5. These studies suggested that the preference of ACSL4 for long-chain polyunsaturated fatty acids can be modified by altering specific amino acid residues.