Department of Biochemical Engineering, University College London, Gower Street, London, WC1E 6BT, UK.
Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK.
Appl Microbiol Biotechnol. 2020 May;104(10):4397-4406. doi: 10.1007/s00253-020-10519-w. Epub 2020 Mar 19.
With increasing interest in the diverse properties of organic acids and their application in synthetic pathways, developing biological tools for producing known and novel organic acids would be very valuable. In such a system, organic acids may be activated as coenzyme A (CoA) esters, then modified by CoA-dependent enzymes, followed by CoA liberation by a broad-acting thioesterase. This study has focused on the identification of suitable thioesterases (TE) for utilisation in such a pathway. Four recombinant hotdog-fold TEs were screened with a range of CoA esters in order to identify a highly active, broad spectrum TE. The TesB-like TE, RpaL, from Rhodopseudomonas palustris was found to be able to use aromatic, alicyclic and both long and short aliphatic CoA esters. Size exclusion chromatography, revealed RpaL to be a monomer of fused hotdog domains, in contrast to the complex quaternary structures found with similar TesB-like TEs. Nonetheless, sequence alignments showed a conserved catalytic triad despite the variation in quaternary arrangement. Kinetic analysis revealed a preference towards short-branched chain CoA esters with the highest specificity towards DL-β-hydroxybutyryl CoA (1.6 × 10 M s), which was found to decrease as the acyl chain became longer and more functionalised. Substrate inhibition was observed with the fatty acyl n-heptadecanoyl CoA at concentrations exceeding 0.3 mM; however, this was attributed to its micellar aggregation properties. As a result of the broad activity observed with RpaL, it is a strong candidate for implementation in CoA ester pathways to generate modified or novel organic acids.
随着人们对有机酸的各种特性及其在合成途径中的应用越来越感兴趣，开发用于生产已知和新型有机酸的生物工具将非常有价值。在这样的系统中，有机酸可以被激活为辅酶 A (CoA) 酯，然后由 CoA 依赖性酶修饰，最后由广谱硫酯酶释放 CoA。本研究主要集中于鉴定适合用于该途径的合适硫酯酶 (TE)。筛选了四种重组热狗折叠 TE，用一系列 CoA 酯进行筛选，以鉴定一种高活性、广谱的 TE。从沼泽红假单胞菌中发现的 TesB 样 TE RpaL 能够使用芳香族、脂环族以及长链和短链脂肪族 CoA 酯。尺寸排阻色谱显示 RpaL 是融合热狗结构域的单体，与类似的 TesB 样 TE 中发现的复杂四级结构形成对比。尽管四级结构存在差异，但序列比对显示出保守的催化三联体。动力学分析显示 RpaL 对短支链 CoA 酯具有偏好性，对 DL-β-羟基丁酰 CoA 的特异性最高（1.6×10^5 M s），随着酰基链变长和功能化，特异性降低。在浓度超过 0.3 mM 时，观察到脂肪酸 n-十七酰 CoA 的底物抑制；然而，这归因于其胶束聚集特性。由于 RpaL 观察到的广泛活性，它是在 CoA 酯途径中生成修饰或新型有机酸的有力候选物。
