Sánchez-Romero Juan José, Olguin Luis F
Laboratorio de Biofisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México, México D. F. 04510, México.
Biochem Biophys Rep. 2015 Aug 7;3:161-168. doi: 10.1016/j.bbrep.2015.08.002. eCollection 2015 Sep.
() is a nitrogen-fixing α-proteobacterium able to biosynthesize the osmoprotectant glycine betaine from choline sulfate through a metabolic pathway that starts with the enzyme choline--sulfatase. This protein seems to be widely distributed in microorganisms and thought to play an important role in their sulfur metabolism. However, only crude extracts with choline sulfatase activity have been studied. In this work, () choline--sulfatase was obtained in a high degree of purity after expression in . Gel filtration and dynamic light scattering experiments showed that the recombinant enzyme exists as a dimer in solution. Using calorimetry, its catalytic activity against its natural substrate, choline--sulfate, gave a =2.7×10 s and a =11.1 mM. For the synthetic substrates -nitrophenyl sulfate and methylumbelliferyl sulfate, the values were 3.5×10 s and 4.3×10 s, with values of 75.8 and 11.8 mM respectively. The low catalytic activity of the recombinant sulfatase was due to the absence of the formylglycine post-translational modification in its active-site cysteine 54. Nevertheless, unmodified () choline--sulfatase is a multiple-turnover enzyme with remarkable catalytic efficiency.
（）是一种固氮α-变形菌，能够通过一条从胆碱硫酸酯酶开始的代谢途径，从硫酸胆碱生物合成渗透保护剂甘氨酸甜菜碱。这种蛋白质似乎在微生物中广泛分布，并被认为在它们的硫代谢中起重要作用。然而，仅对具有胆碱硫酸酯酶活性的粗提物进行了研究。在这项工作中，（）胆碱硫酸酯酶在（表达系统）中表达后获得了高纯度。凝胶过滤和动态光散射实验表明，重组酶在溶液中以二聚体形式存在。使用量热法，其对天然底物硫酸胆碱的催化活性给出了kcat = 2.7×10 s和Km = 11.1 mM。对于合成底物对硝基苯硫酸酯和甲基伞形酮基硫酸酯，kcat值分别为3.5×10 s和4.3×10 s，Km值分别为75.8和11.8 mM。重组硫酸酯酶的低催化活性是由于其活性位点半胱氨酸54缺乏甲酰甘氨酸的翻译后修饰。尽管如此，未修饰的（）胆碱硫酸酯酶是一种具有显著催化效率的多周转酶。
