Liu Weidong, Guo Rey-Ting, Chen Xi, Li Zhe, Gao Xiuzhen, Huang Chun-Hsiang, Wu Qiaqing, Feng Jinhui, Zhu Dunming
Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area, Tianjin 300308 (China).
Chembiochem. 2015 Apr 13;16(6):924-9. doi: 10.1002/cbic.201402632. Epub 2015 Mar 6.
A meso-diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D-amino acids other than its native substrate. Site-directed mutagenesis similar to that carried out on the residues mutated by Vedha-Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D-amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso-diaminopimelate (meso-DAP), D-leucine (D-leu), and 4-methyl-2-oxopentanoic acid (MOPA) were solved. meso-DAP was found in an area outside the catalytic cavity; this suggested a possible two-step substrate-binding mechanism for meso-DAP. D-leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso-diaminopimelate dehydrogenases.
人们发现，破伤风梭菌E88（CtDAPDH）中的中-二氨基庚二酸脱氢酶对其天然底物以外的D-氨基酸活性较低。与Vedha-Peters等人对突变残基所进行的定点诱变相似，诱变产生了一种突变酶，其催化合成D-氨基酸的能力得到了显著提高。解析了无辅基形式以及与中-二氨基庚二酸（meso-DAP）、D-亮氨酸（D-leu）和4-甲基-2-氧代戊酸（MOPA）形成复合物的CtDAPDH突变体的晶体结构。发现meso-DAP位于催化腔外的一个区域；这表明meso-DAP可能存在两步底物结合机制。在CtDAPDH的开放形式中，D-leu和MOPA均与Leu154和Gly155结合，结构分析揭示了突变的中-二氨基庚二酸脱氢酶底物特异性扩展的分子基础。
