Serine palmitoyltransferase (SPTase), the first enzyme of the sphingolipid biosynthesis pathway, produces 3-ketodihydrosphingosine by a Claisen-like condensation/decarboxylation reaction of l-Ser and palmitoyl-CoA (n-C16-CoA). Previous structural analysis of Sphingomonas paucimobilis SPTase (SpSPTase) revealed a dynamic active site loop (RPPATP; amino acids 378-383) in which R378 (underlined) forms a salt bridge with the carboxylic acid group of the PLP : l-Ser external aldimine. We hypothesized that this interaction might play a key role in acyl group substrate selectivity and therefore performed site-saturation mutagenesis at position 378 based on semi-rational design to expand tolerance for shorter acyl-CoA's. The resulting library was initially screened for the reaction between l-Ser and dodecanoyl-CoA (n-C12-CoA). The most interesting mutant (R378 K) was then purified and compared to wild-type SpSPTase against a panel of acyl-CoA's. These data showed that the R378 K substitution shifted the acyl group preference to shorter chain lengths, opening the possibility of using this and other engineered variants for biocatalytic C-C bond-forming reactions.

中文翻译：

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扩大鞘氨醇单胞菌丝氨酸棕榈酰转移酶的酰基辅酶A链长度耐受性的半合理方法

丝氨酸棕榈酰转移酶 (SPTase) 是鞘脂生物合成途径的第一种酶，通过 l-Ser 和棕榈酰辅酶 A (n-C16-CoA) 的 Claisen 样缩合/脱羧反应产生 3-酮二氢鞘氨醇。先前对少动鞘氨醇单胞菌 SPTase (SpSPTase) 的结构分析揭示了一个动态活性位点环 (RPPATP；氨基酸 378-383)，其中 R378（下划线）与 PLP 的羧酸基团形成盐桥：l-Ser 外部醛亚胺。我们假设这种相互作用可能在酰基底物选择性中起关键作用，因此基于半合理设计在 378 位进行位点饱和诱变，以扩大对较短酰基辅酶 A 的耐受性。最初针对 l-Ser 和十二酰-CoA (n-C12-CoA) 之间的反应筛选所得文库。然后纯化最有趣的突变体 (R378 K) 并与针对一组酰基辅酶 A 的野生型 SpSPTase 进行比较。这些数据表明，R378 K 取代将酰基偏好转移到更短的链长，从而开启了使用该变体和其他工程变体进行生物催化 CC 键形成反应的可能性。