Gharib Ghazaleh, Rashid Naeem, Bashir Qamar, Gardner Qura-Tul Ann Afza, Akhtar Muhammad, Imanaka Tadayuki
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan.
School of Biological Sciences, University of Southampton, Southampton, SO16 7PX, UK.
Extremophiles. 2016 Jan;20(1):57-67. doi: 10.1007/s00792-015-0797-3. Epub 2015 Oct 28.
Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90 °C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90 % residual activity even after heating for 6 h in boiling water.
在嗜热栖热放线菌(Pyrobaculum calidifontis)的基因组中发现了两种苹果酸脱氢酶同源物,即Pcal_0564和Pcal_1699。编码Pcal_1699的基因由927个核苷酸组成,对应于一个309个氨基酸的多肽。为了研究Pcal_1699的特性,克隆了其结构基因,在大肠杆菌中表达,并对纯化的基因产物进行了表征。Pcal_1699是一种对NADH具有特异性的酶,在最佳pH值(10)和温度(90°C)下表现出较高的苹果酸脱氢酶活性(886 U/mg)。展开研究表明,即使在最终浓度为8 M时,尿素也不能诱导Pcal_1699完全展开和失活;然而,在4 M盐酸胍存在的情况下,酶结构展开,酶活性完全丧失。热稳定性实验表明,Pcal_1699是迄今为止报道的最耐热的苹果酸脱氢酶,即使在沸水中加热6小时后仍保留超过90%的残余活性。