内消旋-二氨基庚二酸脱氢酶关键氨基酸位点的改造提高对烷基取代2-酮酸的催化活力  

Mutation of a key amino acid residue of meso-diaminopimelate dehydrogenase enhances the catalytic activity toward alkyl substituted 2-keto acids

作　　者：程新宽 陈曦[2] 冯进辉[2] 吴洽庆[2] 朱敦明[2] CHENG Xin-Kuan;CHEN Xi;FENG Jin-Hui;WU Qia-Qing;ZHU Dun-Ming(Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,Tianjin Key Laboratory of Industrial Microbiology,College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China; Tianjin Biocatalysis Technology Engineering Center,National Engineering Laboratory for Industrial Enzymes,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China)

机构地区：[1]天津科技大学生物工程学院,工业发酵微生物教育部重点实验室,天津市工业微生物重点实验室,天津300457 [2]中国科学院天津工业生物技术研究所,工业酶国家工程实验室,天津市生物催化技术工程中心,天津300308

出　　处：《微生物学通报》2020年第7期2119-2127,共9页Microbiology China

基　　金：国家自然科学基金(21778072);天津市科学技术委员会项目(15PTCYSY00020,15PTGCCX00060);天津市教委科研计划(2019KJ239)。

摘　　要：【背景】高效实现D-氨基酸的生物合成一直是人们关注的热点。内消旋-二氨基庚二酸脱氢酶(meso-diaminopimelate dehydrogenase,DAPDH)能够直接催化2-酮酸和氨合成D-氨基酸。【目的】提高DAPDH对烷基取代2-酮酸的催化活力,并解释其催化机制。【方法】以来源于嗜热共生杆菌(Symbiobacteriumthermophilum)的内消旋-二氨基庚二酸脱氢酶(StDAPDH)为模板,在前期结构分析结合被选择位点突变结果的基础上,确定对H227位进行定点饱和突变,并以D-丙氨酸、D-2-氨基丁酸、D-正缬氨酸、D-谷氨酸为底物进行筛选。【结果】获得突变体H227Q和H227N。突变体H227Q对丙酮酸、2-氧代丁酸、2-氧代戊酸、2-酮戊二酸的比活力比野生型分别提高了10.9、11.5、8.6和7.6倍。动力学参数表明,突变体H227Q同时提高了酶对底物的亲和力及催化常数,使其对丙酮酸的催化效率(kcat/Km)相较于野生型提高了9.4倍。利用分子模拟技术分析突变体H227Q与产物氨基酸之间的相互作用表明,227位的谷氨酰胺通过与氨基酸的羧酸形成氢键,使得氨基酸产物Cα上的氢和辅酶烟酰胺环C4原子之间的距离缩短。【结论】利用定向进化技术提高DAPDH对烷基取代2-酮酸的催化活力,有助于开发新型的高效生物催化剂,这些工作也为下一步继续进行更具挑战性的D-氨基酸研究提供了基础。[Background] Efficient biosynthesis of D-amino acids is highly desired. Meso-diaminopimelate dehydrogenase(DAPDH) synthesizes D-amino acids from 2-keto acids and ammonia. [Objective] To increase the catalytic activity against the alkyl substituted 2-keto acids. [Methods] Based on structural analysis and mutation results from previously selected sites, the saturation mutagenesis was carried out at the amino acid residue H227 of DAPDH from Symbiobacterium thermophilum(StDAPDH). The resulting mutant library was subjected to screening using D-alanine, D-2-aminobutyric acid, D-norvaline, and D-glutamic acid as substrates. [Results] The mutants H227 Q and H227 N were obtained. Mutant H227 Q was found to have 10.9-, 11.5-, 8.6-and 7.6-folds improved enzyme activity toward pyruvic acid, 2-oxobutyric acid, 2-oxovaleric acid and 2-ketoglutaric acid, respectively, compared to that of wild-type enzyme. The kinetic parameters indicated that mutant H227 Q increased the turnover number of the enzyme and the affinity of the enzyme for the substrate simultaneously, so that the catalytic efficiency(kcat/Km) of pyruvic acid was 9.4 folds higher than that of wild-type enzyme. Molecular modeling analysis of interaction between mutant H227 Q and product amino acid, indicating that glutamine at position H227 forms a hydrogen bond with the carboxylic acid of the amino acid, so that the distance between the α-hydrogen atom of product amino acid and C4 of coenzyme nicotinamide ring was shortened. [Conclusion] Directed evolution technology has been successfully used to improve the catalytic activity of DADPH for alkyl-substituted 2-keto acids, which is helpful for the development of new high-efficiency biocatalysts. These efforts also provide guidance for our future engineering of this enzyme about more challenging D-amino acids.

关 键 词：烷基取代2-酮酸 内消旋-二氨基庚二酸脱氢酶 饱和突变 分子对接 D-氨基酸 

分 类 号：Q936[生物学—微生物学]