Dale S, Arró M, Becerra B, Morrice N G, Boronat A, Hardie D G, Ferrer A
Biochemistry Department, The University, Dundee, UK.
Eur J Biochem. 1995 Oct 15;233(2):506-13. doi: 10.1111/j.1432-1033.1995.506_2.x.
The catalytic domain of 3-hydroxy-3-methylglutaryl-CoA reductase isoform 1 (HMGR1cd) from Arabidopsis thaliana has been expressed in Escherichia coli in a catalytically active form and purified. The high efficiency of the bacterial expression system together with the simplicity of the purification procedure used in this study resulted in the attainment of large quantities of pure enzyme (about 5 mg/l culture) with a final specific activity of up to 17 U/mg. This specific activity is higher than that reported to date for any 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) purified from a plant source. HMGR1cd activity was completely blocked by the HMGR inhibitor mevinolin (IC50 = 12.5 nM). No significant differences were observed between the Km values of HMGR1cd for NADPH (71 +/- 7 microM) and (S)-3-hydroxy-3-methylglutaryl-CoA (8.3 +/- 1.5 microM) and those of pure HMGR preparations obtained from different plant sources. The purified HMGR1cd was reversibly inactivated by phosphorylation at a single site by Brassica oleracea HMGR kinase A, which is functionally related to the mammalian AMP-activated protein kinase. The site of phosphorylation is Ser577 in the complete sequence of A. thaliana HMGR1. The results in this paper represent the first evidence that a higher plant HMGR is regulated by direct phosphorylation, at least in a cell-free system. Our results also reinforce the view that the AMP-activated protein kinase/SNF1 family is an ancient and highly conserved protein kinase system.
拟南芥3-羟基-3-甲基戊二酰辅酶A还原酶同工型1（HMGR1cd）的催化结构域已在大肠杆菌中以具有催化活性的形式表达并纯化。本研究中细菌表达系统的高效率以及所采用纯化程序的简单性，使得能够获得大量的纯酶（约5 mg/升培养物），最终比活性高达17 U/mg。该比活性高于迄今为止从植物来源纯化得到的任何3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR）所报道的比活性。HMGR1cd的活性被HMGR抑制剂美伐他汀完全阻断（IC50 = 12.5 nM）。在HMGR1cd对NADPH（71±7 μM）和（S）-3-羟基-3-甲基戊二酰辅酶A（8.3±1.5 μM）的Km值与从不同植物来源获得的纯HMGR制剂的Km值之间未观察到显著差异。纯化的HMGR1cd被与哺乳动物AMP激活的蛋白激酶功能相关的甘蓝型油菜HMGR激酶A在单个位点磷酸化而可逆失活。磷酸化位点是拟南芥HMGR1完整序列中的Ser577。本文的结果代表了首个证据，即高等植物HMGR至少在无细胞系统中受直接磷酸化调控。我们的结果还强化了这样一种观点，即AMP激活的蛋白激酶/SNF1家族是一个古老且高度保守的蛋白激酶系统。
