Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol.
Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol.
复制标题
DOI：
10.1016/j.jbiotec.2012.08.008
复制
发表时间：
2012-12-15
影响因子：
4.1
通讯作者：
Arnold, Frances H.
中科院分区：
文献类型：
--
作者：
Liu, Xiang;Bastian, Sabine;Snow, Christopher D.;Brustad, Eric M.;Saleski, Tatyana E.;Xu, Jian-He;Meinhold, Peter;Arnold, Frances H.
关键词：
我们已经确定了来自乳酸乳球菌的依赖于NADH的醇脱氢酶LlAdhA及其实验室进化的变体LlAdhARE 1的X射线晶体结构，分别在1.9 μ m和2.5 μ m分辨率。含有三个氨基酸突变（Y50 F、I212 T和L264 V）的LlAdhARE 1被工程化以增加从异丁醛（2-甲基丙醛）微生物生产异丁醇（2-甲基丙-1-醇）。LlAdhA和LlAdhARE 1的结构比较表明，对异丁醛的增强活性源于蛋白质的活性位点大小、疏水性和底物接近性的增加。进一步的结构导向诱变产生了四重突变体（Y50 F/N110 S/I212 T/L264 V），其对异丁醛的KM比野生型LlAdhA低约17倍，催化效率（kcat/KM）比野生型LlAdhA高约160倍。结合详细的结构信息和定向进化，我们已经实现了非天然醇脱氢酶活性的显着改善，这将有助于从可再生资源中生产下一代燃料，如异丁醇。
We have determined the X-ray crystal structures of the NADH-dependent alcohol dehydrogenase LlAdhA from Lactococcus lactis and its laboratory-evolved variant LlAdhARE1 at 1.9 Å and 2.5 Å resolution, respectively. LlAdhARE1, which contains three amino acid mutations (Y50F, I212T, and L264V), was engineered to increase the microbial production of isobutanol (2-methylpropan-1-ol) from isobutyraldehyde (2-methylpropanal). Structural comparison of LlAdhA and LlAdhARE1 indicates that the enhanced activity on isobutyraldehyde stems from increases in the protein’s active site size, hydrophobicity, and substrate access. Further structure-guided mutagenesis generated a quadruple mutant (Y50F/N110S/I212T/L264V), whose KM for isobutyraldehyde is ~17-fold lower and catalytic efficiency (kcat/KM) is ~160-fold higher than wild-type LlAdhA. Combining detailed structural information and directed evolution, we have achieved significant improvements in non-native alcohol dehydrogenase activity that will facilitate the production of next-generation fuels such as isobutanol from renewable resources.
登录
查看更多内容
DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
Evans, P
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1073/pnas.96.19.10689
发表时间：
1999-09-14
影响因子：
11.1
作者：
Bornberg-Bauer, E;Chan, HS
通讯作者：
Chan, HS
影响因子：
8
作者：
Eklund, H.;Ramaswamy, S.
通讯作者：
Ramaswamy, S.
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH