Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012, USA.
J Biol Chem. 2013 Jun 21;288(25):18473-83. doi: 10.1074/jbc.M113.466912. Epub 2013 Apr 26.
Mycobacterium tuberculosis has a group of 34 FadD proteins that belong to the adenylate-forming superfamily. They are classified as either fatty acyl-AMP ligases (FAALs) or fatty acyl-CoA ligases based on sequence analysis. FadD10, involved in the synthesis of a virulence-related lipopeptide, was mis-annotated as a fatty acyl-CoA ligase; however, it is in fact a FAAL that transfers fatty acids to an acyl carrier protein (Rv0100). In this study, we have determined the structures of FadD10 in both the apo-form and the complexed form with dodecanoyl-AMP, where we see for the first time an adenylate-forming enzyme that does not adopt a closed conformation for catalysis. Indeed, this novel conformation of FadD10, facilitated by its unique inter-domain and intermolecular interactions, is critical for the enzyme to carry out the acyl transfer onto Rv0100 rather than coenzyme A. This contradicts the existing model of FAALs that rely on an insertion motif for the acyltransferase specificity and thus makes FadD10 a new type of FAAL. We have also characterized the fatty acid preference of FadD10 through biological and structural analyses, and the data indicate long chain saturated fatty acids as the biological substrates of the enzyme.
结核分枝杆菌有一组 34 种 FadD 蛋白，属于腺苷酸形成超家族。根据序列分析，它们被分类为脂肪酰基-AMP 连接酶（FAALs）或脂肪酰基辅酶 A 连接酶。参与合成与毒力相关的脂肽的 FadD10 被错误注释为脂肪酰基辅酶 A 连接酶；然而，它实际上是一种将脂肪酸转移到酰基载体蛋白（Rv0100）的 FAAL。在这项研究中，我们确定了 FadD10 在apo 形式和与十二烷酰基-AMP 复合形式的结构，我们首次看到一种不采用封闭构象进行催化的腺苷酸形成酶。事实上，这种 FadD10 的新颖构象，由其独特的域间和分子间相互作用促进，对于酶将酰基转移到 Rv0100 而不是辅酶 A 至关重要。这与依赖插入模体来确定酰基转移酶特异性的现有 FAAL 模型相矛盾，因此使 FadD10 成为一种新型的 FAAL。我们还通过生物学和结构分析表征了 FadD10 的脂肪酸偏好性，数据表明长链饱和脂肪酸是该酶的生物底物。
