Sutherlin Autumn, Rodwell Victor W
Department of Biochemistry, Purdue University, 175 South University Street, West Lafayette, Indiana 47907-2063, USA.
Biotechnol Bioeng. 2004 Aug 20;87(4):546-51. doi: 10.1002/bit.20157.
The five-carbon metabolic intermediate isopentenyl diphosphate constitutes the basic building block for the biosynthesis of all isoprenoids in all forms of life. Two distinct pathways lead from amphibolic intermediates to isopentenyl diphosphate. The Gram-positive cocci and certain other pathogenic bacteria employ exclusively the mevalonate pathway, a set of six enzyme-catalyzed reactions that convert 3 mol of acetyl-CoA to 1 mol each of carbon dioxide and isopentenyl diphosphate. The survival of the Gram-positive cocci requires a fully functional set of mevalonate pathway enzymes. These enzymes therefore represent potential targets of inhibitors that might be employed as antibiotics directed against multidrug-resistant strains of certain bacterial pathogens. A rapid throughput, bioreactor-based assay to assess the effects of potential inhibitors on several enzymes simultaneously should prove useful for the survey of candidate inhibitors. To approach this goal, and as a proof of concept, we employed enzymes from the Gram-positive pathogen Enterococcus faecalis. Purified recombinant enzymes that catalyze the first three reactions of the mevalonate pathway were immobilized in two kinds of continuous flow enzyme bioreactors: a classical hollow fiber bioreactor and an immobilized plug flow bioreactor that exploited a novel method of enzyme immobilization. Both bioreactor types employed recombinant acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase from E. faecalis to convert acetyl-CoA to mevalonate, the central intermediate of the mevalonate pathway. Reactor performance was monitored continuously by spectrophotometric measurement of the concentration of NADPH in the reactor effluent. Additional potential applications of an Ni(++) affinity support bioreactor include using recombinant enzymes from extremophiles for biosynthetic applications. Finally, linking a Ni(++) affinity support bioreactor to an HPLC-mass spectrometer would provide an experimental and pedagogical tool for study of metabolite flux and pool sizes of intermediates to model regulation in intact cells.
五碳代谢中间体异戊烯基二磷酸是所有生命形式中所有类异戊二烯生物合成的基本构件。从两用代谢中间体到异戊烯基二磷酸有两条不同的途径。革兰氏阳性球菌和某些其他致病细菌仅采用甲羟戊酸途径，这是一组六个酶催化反应，将3摩尔乙酰辅酶A转化为1摩尔二氧化碳和1摩尔异戊烯基二磷酸。革兰氏阳性球菌的存活需要一套功能齐全的甲羟戊酸途径酶。因此，这些酶代表了抑制剂的潜在靶点，这些抑制剂可作为针对某些细菌病原体多药耐药菌株的抗生素。一种基于生物反应器的快速通量测定法，用于同时评估潜在抑制剂对几种酶的影响，对于筛选候选抑制剂应是有用的。为了实现这一目标，并作为概念验证，我们使用了革兰氏阳性病原体粪肠球菌的酶。催化甲羟戊酸途径前三个反应的纯化重组酶被固定在两种连续流动酶生物反应器中：一种经典的中空纤维生物反应器和一种利用新型酶固定方法的固定化活塞流生物反应器。两种生物反应器类型都使用来自粪肠球菌的重组乙酰乙酰辅酶A硫解酶、HMG辅酶A合酶和HMG辅酶A还原酶，将乙酰辅酶A转化为甲羟戊酸，这是甲羟戊酸途径的中心中间体。通过分光光度法连续监测反应器流出物中NADPH的浓度来监测反应器性能。Ni(++)亲和支持生物反应器的其他潜在应用包括使用来自极端微生物的重组酶进行生物合成应用。最后，将Ni(++)亲和支持生物反应器与高效液相色谱-质谱仪连接，将为研究完整细胞中代谢物通量和中间体库大小以模拟调节提供一种实验和教学工具。
