Mutational analysis of a fatty acyl-coenzyme A synthetase signature motif identifies seven amino acid residues that modulate fatty acid substrate specificity
Mutational analysis of a fatty acyl-coenzyme A synthetase signature motif identifies seven amino acid residues that modulate fatty acid substrate specificity
复制标题
DOI：
10.1074/jbc.272.8.4896
复制
发表时间：
1997-02-21
影响因子：
4.8
通讯作者：
DiRusso, CC
中科院分区：
文献类型：
--
作者：
Black, PN;Zhang, Q;DiRusso, CC
脂肪酰辅酶A合成酶(脂肪酸：辅酶A连接酶，AMP形成；EC 6.2.1.3)通过两步过程催化脂肪酰辅酶A的形成，该过程通过焦磷酸的水解进行。在大肠杆菌中，该酶在长链脂肪酸(C12-C18)的摄取和全球转录调节因子FadR的调节中起着关键作用，大肠杆菌脂肪酰基-CoA合成酶与原核和真核脂肪酰基-CoA合成酶家族具有显著的氨基酸相似性和同一性，表明共同的祖先，在这方面最值得注意的是26个氨基酸的共同序列，DGWLHTGDIGXWX-PXGXLKIIDRKEE，对于所有可获得序列信息的脂肪酰基-CoA合成酶，在这个共识中，比较12个脂肪酰基-CoA合成酶中的8个不变和13个高度保守的氨基酸残基我们认为该序列代表脂肪酰辅酶A合成酶的签名基序(FAGS签名基序)。大肠杆菌脂肪酰辅酶A合成酶的这个区域(431)NGWLHTGDIAVMDEEGFL-RIVDRKK(455)包含17个氨基酸残基，它们与FAGS特征基序相同或高度保守。构建了18个与该基序对应的脂肪酰辅酶A合成酶结构基因(FADD)的定点突变，以评估该酶的该区域对催化活性的贡献。根据脂肪酸的生长特性、使用细胞提取物的酶活性以及使用纯化的野生型和突变形式的酶的研究，鉴定了三种不同类型的突变：1)导致野生型或接近野生型脂肪酰辅酶A合成酶活性的突变；2)酶活性很低或没有酶活性的突变；3)那些导致脂肪酸链长专一性降低和改变的突变体，在所鉴定的18个突变体中，有7个属于第三类，我们认为FAGS特征基序是催化活性所必需的，部分功能是促进脂肪酸链长专一性，从而可能构成酶内脂肪酸结合部位的一部分。
Fatty acyl-CoA synthetase (fatty acid:CoA ligase, AMP-forming; EC 6.2.1.3) catalyzes the formation of fatty acyl-CoA by a two-step process that proceeds through the hydrolysis of pyrophosphate. In Escherichia coli this enzyme plays a pivotal role in the uptake of long chain fatty acids (C12-C18) and in the regulation of the global transcriptional regulator FadR, The E. coli fatty acyl-CoA synthetase has remarkable amino acid similarities and identities to the family of both prokaryotic and eukaryotic fatty acyl-CoA synthetases, indicating a common ancestry, Most notable in this regard is a 26-amino acid consensus sequence, DGWLHTGDIGXWX-PXGXLKIIDRKEE, common to all fatty acyl-CoA synthetases for which sequence information is available, Within this consensus are 8 invariant and 13 highly conserved amino acid residues in the 12 fatty acyl-CoA synthetases compared, We propose that this sequence represents the fatty acyl-CoA synthetase signature motif (FAGS signature motif). This region of fatty acyl-CoA synthetase from E. coli, (431)NGWLHTGDIAVMDEEGFL-RIVDRKK(455), contains 17 amino acid residues that are either identical or highly conserved to the FAGS signature motif. Eighteen site directed mutations within the fatty acyl-CoA synthetase structural gene (fadD) corresponding to this motif were constructed to evaluate the contribution of this region of the enzyme to catalytic activity, Three distinct classes of mutations were identified on the basis of growth characteristics on fatty acids, enzymatic activities using cell extracts, and studies using purified wild-type and mutant forms of the enzyme: 1) those that resulted in either wild-type or nearly wild-type fatty acyl-CoA synthetase activity profiles; 2) those that had little or no enzyme activity; and 3) those that resulted in lowering and altering fatty acid chain length specificity, Among the 18 mutants characterized, 7 fall in the third class, We propose that the FAGS signature motif is essential for catalytic activity and functions in part to promote fatty acid chain length specificity and thus may compose part of the fatty acid binding site within the enzyme.