Finnegan Steffan, Gadda Giovanni
Departments of Chemistry and Biology, The Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia 30302-4098, USA.
Biochemistry. 2008 Dec 30;47(52):13850-61. doi: 10.1021/bi801424p.
The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions. This is relevant for the genetic engineering of crops with economic interest that do not naturally possess efficient pathways for the synthesis of glycine betaine and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, the best characterized choline-oxidizing enzyme is the flavin-dependent choline oxidase from Arthrobacter globiformis, for which structural, mechanistic, and biochemical data are available. Here, we have replaced a hydrophobic residue (Val464) lining the active site cavity close to the N(5) atom of the flavin with threonine or alanine to investigate its role in the reaction of choline oxidation catalyzed by choline oxidase. The reductive half-reactions of the enzyme variants containing Thr464 or Ala464 were investigated using substrate and solvent kinetic isotope effects, solvent viscosity effects, and proton inventories. Replacement of Val464 with threonine or alanine uncovered a kinetically slow equilibrium between a catalytically incompetent form of enzyme and an active species that can efficiently oxidize choline. In both variants, the active form of enzyme shows a decreased rate of hydroxyl proton abstraction from the alcohol substrate, with minimal changes in the subsequent rate of hydride ion transfer to the flavin. This study therefore establishes that a hydrophobic residue not directly participating in catalysis plays important roles in the reaction of choline oxidation catalyzed by choline oxidase.
胆碱酶促氧化生成甘氨酸甜菜碱备受关注，因为生物体在应激条件下会在细胞内积累甘氨酸甜菜碱。这对于具有经济价值的作物的基因工程具有重要意义，这些作物天然不具备高效合成甘氨酸甜菜碱的途径，同时对于开发针对人类病原体中甘氨酸甜菜碱生物合成途径的潜在药物也具有重要意义。迄今为止，表征最充分的胆碱氧化酶是来自球形节杆菌的黄素依赖性胆碱氧化酶，已有关于其结构、作用机制和生化数据。在此，我们用苏氨酸或丙氨酸取代了位于活性位点腔靠近黄素N(5)原子处的一个疏水残基（Val464），以研究其在胆碱氧化酶催化的胆碱氧化反应中的作用。使用底物和溶剂动力学同位素效应、溶剂粘度效应和质子丰度研究了含有Thr464或Ala464的酶变体的还原半反应。用苏氨酸或丙氨酸取代Val464揭示了一种催化无活性形式的酶与一种能够有效氧化胆碱的活性物种之间在动力学上缓慢的平衡。在这两种变体中，酶的活性形式显示从醇底物中夺取羟基质子的速率降低，而随后氢负离子转移到黄素的速率变化最小。因此，本研究确定了一个不直接参与催化的疏水残基在胆碱氧化酶催化的胆碱氧化反应中起重要作用。
