Jia Kaimin, Cao Ruikai, Hua Duy H, Li Ping
Department of Chemistry, Kansas State University , Manhattan, Kansas 66506, United States.
Biomacromolecules. 2016 Apr 11;17(4):1477-85. doi: 10.1021/acs.biomac.6b00082. Epub 2016 Mar 22.
Polyhydroxyalkanoates (PHAs) are carbon and energy storage polymers produced by a variety of microbial organisms under nutrient-limited conditions. They have been considered as an environmentally friendly alternative to oil-based plastics due to their renewability, versatility, and biodegradability. PHA synthase (PhaC) plays a central role in PHA biosynthesis, in which its activity and substrate specificity are major factors in determining the productivity and properties of the produced polymers. However, the effects of modifying the substrate side chain are not well understood because of the difficulty to accessing the desired analogues. In this report, a series of 3-(R)-hydroxyacyl coenzyme A (HACoA) analogues were synthesized and tested with class I synthases from Chromobacterium sp. USM2 (PhaCCs and A479S-PhaCCs) and Caulobacter crescentus (PhaCCc) as well as class III synthase from Allochromatium vinosum (PhaECAv). It was found that, while different PHA synthases displayed distinct preference with regard to the length of the alkyl side chains, they could withstand moderate side chain modifications such as terminal unsaturated bonds and the azide group. Specifically, the specific activity of PhaCCs toward propynyl analogue (HHxyCoA) was only 5-fold less than that toward the classical substrate HBCoA. The catalytic efficiency (kcat/Km) of PhaECAv toward azide analogue (HABCoA) was determined to be 2.86 × 10(5) M(-1) s(-1), which was 6.2% of the value of HBCoA (4.62 × 10(6) M(-1) s(-1)) measured in the presence of bovine serum albumin (BSA). These side chain modifications may be employed to introduce new material functions to PHAs as well as to study PHA biogenesis via click-chemistry, in which the latter remains unknown and is important for metabolic engineering to produce PHAs economically.
聚羟基脂肪酸酯（PHA）是多种微生物在营养受限条件下产生的碳和能量储存聚合物。由于其可再生性、多功能性和生物可降解性，它们被认为是石油基塑料的环保替代品。PHA合酶（PhaC）在PHA生物合成中起核心作用，其活性和底物特异性是决定所生产聚合物的生产率和性能的主要因素。然而，由于难以获得所需的类似物，修饰底物侧链的影响尚未得到很好的理解。在本报告中，合成了一系列3-(R)-羟基酰基辅酶A（HACoA）类似物，并用来自嗜色杆菌属USM2（PhaCCs和A479S-PhaCCs）和新月柄杆菌（PhaCCc）的I类合酶以及来自嗜盐红菌（PhaECAv）的III类合酶进行了测试。结果发现，虽然不同的PHA合酶对烷基侧链的长度表现出不同的偏好，但它们能够承受适度的侧链修饰，如末端不饱和键和叠氮基团。具体而言，PhaCCs对丙炔基类似物（HHxyCoA）的比活性仅比对经典底物HBCoA的比活性低5倍。在牛血清白蛋白（BSA）存在下测得，PhaECAv对叠氮类似物（HABCoA）的催化效率（kcat/Km）为2.86×10(5) M(-1) s(-1)，是HBCoA（4.62×10(6) M(-1) s(-1)）值的6.2%。这些侧链修饰可用于为PHA引入新的材料功能，以及通过点击化学研究PHA生物合成，其中后者仍然未知且对经济地生产PHA的代谢工程很重要。
