Department of Molecular Biology, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan.
Biochemistry. 2011 Oct 11;50(40):8583-93. doi: 10.1021/bi200925f. Epub 2011 Sep 19.
Although enzymes of thermophilic organisms are often very resistant to thermal denaturation, they are usually less active than their mesophilic or psychrophilic homologues at moderate or low temperatures. To explore the structural features that would improve the activity of a thermophilic enzyme at less than optimal temperatures, we randomly mutated the DNA of single-site mutants of the thermostable Thermus thermophilus 3-isopropylmalate dehydrogenase that already had improved low-temperature activity and selected for additional improved low-temperature activity. A mutant (Ile279 → Val) with improved low-temperature activity contained a residue that directly interacts with the adenine of the coenzyme NAD(+), suggesting that modulation of the coenzyme-binding pocket's volume can enhance low-temperature activity. This idea was further supported by a saturation mutagenesis study of the two codons of two other residues that interact with the adenine. Furthermore, a similar type of amino acid substitution also improved the catalytic efficiency of another thermophilic dehydrogenase, T. thermophilus lactate dehydrogenase. Steady-state kinetic experiments showed that the mutations all favorably affected the catalytic turnover numbers. Thermal stability measurements demonstrated that the mutants remain very resistant to heat. Calculation of the energetic contributions to catalysis indicated that the increased turnover numbers are the result of destabilized enzyme-substrate-coenzyme complexes. Therefore, small changes in the side chain volumes of coenzyme-binding residues improved the catalytic efficiencies of two thermophilic dehydrogenases while preserving their high thermal stabilities and may be a way to improve low-temperature activities of dehydrogenases in general.
尽管嗜热生物的酶通常对热变性非常耐受，但它们在中温和低温下的活性通常低于其中温或低温的同源物。为了探索能够提高嗜热酶在非最佳温度下活性的结构特征，我们随机突变了已经具有改善低温活性的耐热 Thermus thermophilus 3-异丙基苹果酸脱氢酶单点突变体的 DNA，并选择了具有额外改善低温活性的突变体。一个具有改善低温活性的突变体（Ile279 → Val）含有一个与辅酶 NAD（+）的腺嘌呤直接相互作用的残基，表明辅酶结合口袋体积的调节可以增强低温活性。对与腺嘌呤相互作用的两个其他残基的两个密码子进行饱和诱变研究进一步支持了这一观点。此外，类似类型的氨基酸取代也提高了另一种嗜热脱氢酶 T. thermophilus 乳酸脱氢酶的催化效率。稳态动力学实验表明，这些突变都有利于提高催化周转率。热稳定性测量表明，突变体仍然对热非常耐受。对催化作用的能量贡献的计算表明，增加的周转率是由于酶-底物-辅酶复合物的不稳定造成的。因此，辅酶结合残基侧链体积的微小变化提高了两种嗜热脱氢酶的催化效率，同时保持了它们的高热稳定性，并且可能是提高一般脱氢酶低温活性的一种方法。
