Inactivation of GDP-mannose dehydrogenase from Pseudomonas aeruginosa by penicillic acid identifies a critical active site loop
Inactivation of GDP-mannose dehydrogenase from Pseudomonas aeruginosa by penicillic acid identifies a critical active site loop
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DOI：
10.1016/j.abb.2005.06.028
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发表时间：
2005-09-15
影响因子：
3.9
通讯作者：
Tipton, PA
中科院分区：
文献类型：
--
作者：
Kimmel, JL;Tipton, PA
致病菌铜绿假单胞菌合成海藻酸盐作为感染过程中产生的一组毒力因子之一。甘露糖脱氢酶催化海藻酸盐生物合成的承诺步骤。研究表明，青霉素酸是gdp -甘露糖脱氢酶的不可逆失活剂，在pH 8.0下，其失活速率常数为0.39 +/- 0.01 mM(-1) min(-1)，且不表现出饱和行为。gdp -甘露糖提供了部分失活保护，而其他底物NAD（+）则没有。GMP和NAD（+）一起提供完全的抗失活保护。质谱分析证实该酶在多个半胱氨酸残基上被青霉素酸烷基化，包括Cys 213、Cys 246和活性位点半胱氨酸Cys 268。然而，失活率的pH依赖性表明，单个半胱氨酸残基的烷基化足以使酶失活。C268A突变蛋白也容易被青霉酸灭活。NAD（+）和GMP的存在对cys246和cys268有部分保护作用，对cys213有几乎完全的保护作用。cys213位于gdp -甘露糖结合袋的一部分螺旋上，并与Asn 252形成氢键。Asn 252位于gdp -甘露糖周围的一个环上。C213A突变酶的V-(max)值是野生型酶的1.8倍，这表明cys213和Asn 252之间的相互作用有助于在催化过程中保持环的位置，而打开环释放产物在一定程度上是限速的。cys246紧邻gdp -甘露糖结合环，其烷基化也可能干扰环的运动。(c) 2005爱思唯尔公司版权所有。
The pathogenic bacterium Pseudomonas aeruginosa synthesizes alginate as one of a group of virulence factors that are produced during infections. The enzyme GDP-mannose dehydrogenase catalyzes the committed step in alginate biosynthesis. We show here that penicillic acid is an irreversible inactivator of GDP-mannose dehydrogenase, Inactivation occurs with it rate constant of 0.39 +/- 0.01 mM(-1) min(-1) at pH 8.0, and does not exhibit saturation behavior. Partial protection from inactivation is afforded by GDP-mannose, but not by the other substrate, NAD(+). GMP and NAD(+) together provide complete protection against inactivation. Analysis by mass spectrometry confirmed that the enzyme is alkylated at multiple cysteine residues by penicillic acid, including Cys 213, Cys 246, and the active site cysteine, Cys 268. However, the pH dependence of the inactivation rate suggested that alkylation of a single cysteine residue is sufficient to inactivate the enzyme. The C268A mutant protein was also susceptible to inactivation by penicillic acid. The presence of NAD(+) and GMP provided partial protection of Cys 246 and Cys 268, and almost complete protection of Cys 213. Cys 213 is located on a helix that forms part of the binding pocket for GDP-mannose, and forms a hydrogen bond with Asn 252. Asn 252 is located on a loop that surrounds GDP-mannose. The C213A mutant enzyme exhibits a V-(max) that is 1.8-fold greater than the wild-type enzyme, suggesting that the interaction between Cys 213 and Asn 252 helps to hold the loop in place during catalysis, and that opening the loop to release product is partially rate-limiting. Cys 246 is adjacent to the GDP-mannose binding loop, and its alkylation may also interfere with loop movement. (c) 2005 Elsevier Inc. All rights reserved.