Wang Yi, Li Yue, Wu Yan, Yan Honggao
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
FEBS J. 2007 May;274(9):2240-52. doi: 10.1111/j.1742-4658.2007.05761.x. Epub 2007 Mar 27.
Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8-dihydro-D-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-L-monapterin (DHMP). Whether the epimerization reaction uses the same reaction intermediate as the aldol reaction or the deprotonation and reprotonation of C2' of DHNP has been investigated by NMR analysis of the reaction products in a D2O solvent. No deuteration of C2' was observed for the newly formed DHMP. This result strongly suggests that the epimerization reaction uses the same reaction intermediate as the aldol reaction. In contrast with an earlier observation, the DHNA-catalyzed reaction is reversible, which also supports a nonstereospecific retroaldol/aldol mechanism for the epimerization reaction. The binding and catalytic properties of DHNAs from both Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA) were determined by equilibrium binding and transient kinetic studies. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism was determined for both SaDHNA and EcDHNA. The results show that the two enzymes have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them.
二氢新蝶呤醛缩酶（DHNA）催化7,8 - 二氢 - D - 新蝶呤（DHNP）裂解形成6 - 羟甲基 - 7,8 - 二氢蝶呤（HP）和乙醇醛，以及催化DHNP差向异构化形成7,8 - 二氢 - L - 单蝶呤（DHMP）。通过在D2O溶剂中对反应产物进行核磁共振分析，研究了差向异构化反应是否与醛缩反应使用相同的反应中间体，或者DHNP的C2'去质子化和再质子化情况。对于新形成的DHMP，未观察到C2'的氘代。这一结果有力地表明差向异构化反应与醛缩反应使用相同的反应中间体。与早期观察结果相反，DHNA催化的反应是可逆的，这也支持了差向异构化反应的非立体特异性逆醛缩/醛缩机制。通过平衡结合和瞬态动力学研究确定了金黄色葡萄球菌（SaDHNA）和大肠杆菌（EcDHNA）中DHNA的结合和催化特性。根据统一的动力学机制，确定了SaDHNA和EcDHNA醛缩反应和差向异构化反应的完整动力学常数集。结果表明，这两种酶具有显著不同的结合和催化特性，这与它们之间显著的序列差异一致。
