Refolding, characterization and crystal structure of (S)-malate dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix
Refolding, characterization and crystal structure of (S)-malate dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix
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DOI：
10.1016/j.bbapap.2009.06.014
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发表时间：
2009-10-01
影响因子：
3.2
通讯作者：
Ohshima, Toshihisa
中科院分区：
文献类型：
--
作者：
Kawakami, Ryushi;Sakuraba, Haruhiko;Ohshima, Toshihisa
从一株嗜热嗜氧菌Aeropyrum Pernix的粗提液中发现了酒石酸的氧化活性，该酶被鉴定为(S)-苹果酸脱氢酶，该酶在大肠杆菌中产生时，主要以非活性的包涵体形式存在。包涵体在6M盐酸胍中溶解，通过稀释变性剂逐渐复性为活性酶。纯化的重组酶由4个相同的亚基组成，分子质量约为110 kDa。与其他来源的MdHS不同，NADP是(S)-苹果酸氧化的首选辅酶，并且该酶很容易催化(2S，3S)-酒石酸和(2S，3R)-酒石酸的氧化。从高温古生菌甲烷钙球菌和黄褐古菌中也观察到酒石酸盐氧化活性，表明这些高温古菌与其底物结合松散。复性后的Pernix MDH也被结晶，确定了其结构，分辨率为2.9埃。其总体结构与金线虫、橙色绿僵菌、弧菌和微小隐孢子虫相似，均方根值在1.4埃到2.1埃之间。与以前的报道一致，53位的Ala负责辅酶专一性，下一个残基Arg对NADP结合很重要。结构比较表明，Pernix MDH的超热稳定性可能是由于其较小的空腔体积以及较多的离子对和离子对网络，但热稳定性的分子策略可能因每种酶而异。(C)2009爱思唯尔B.V.保留所有权利。
Tartrate oxidation activity was found in the crude extract of an aerobic hyperthermophilic archaeon Aeropyrum pernix, and the enzyme was identified as (S)-malate dehydrogenase (MDH), which, when produced in Escherichia coli, was mainly obtained as an inactive inclusion body. The inclusion body was dissolved in 6 M guanidine-HCl and gradually refolded to the active enzyme through dilution of the denaturant. The purified recombinant enzyme consisted of four identical subunits with a molecular mass of about 110 kDa. NADP was preferred as a coenzyme over NAD for (S)-malate oxidation and, unlike MDHs from other sources, this enzyme readily catalyzed the oxidation of (2S,3S)-tartrate and (2S,3R)-tartrate. The tartrate oxidation activity was also observed in MDHs from the hyperthermophilic archaea Methanocaldococcus jannaschii and Archaeoglobus fulgidus, suggesting these hyperthermophilic MDHs loosely bind their substrates. The refolded A. pernix MDH was also crystallized, and the structure was determined at a resolution of 2.9 angstrom. Its overall structure was similar to those of the M. jannaschii, Chloraflexus aurantiacus, Chlorobium vibrioforme and Cryptosporidium parvum [lactate dehydrogenase-like] MDHs with root-mean-square-deviation values between 1.4 and 2.1 angstrom. Consistent with earlier reports, Ala at position 53 was responsible for coenzyme specificity, and the next residue, Arg, was important for NADP binding. Structural comparison revealed that the hyperthermostability of the A. pernix MDH is likely attributable to its smaller cavity volume and larger numbers of ion pairs and ion-pair networks, but the molecular strategy for thermostability may be specific for each enzyme. (C) 2009 Elsevier B.V. All rights reserved.