Heterologous expression, purification, and characterization of an L-ornithine N5-hydroxylase involved in pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa
Heterologous expression, purification, and characterization of an L-ornithine N5-hydroxylase involved in pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa
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DOI：
10.1128/jb.00949-06
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发表时间：
2006-10-01
影响因子：
3.2
通讯作者：
Seah, Stephen Y. K.
中科院分区：
文献类型：
--
作者：
Ge, Li;Seah, Stephen Y. K.
铜绿假单胞菌是一种机会性病原体，可产生铁载体pyoverdine，使其能够从宿主那里获取必需的营养铁。 pyoverdine 中铁螯合异羟肟酸官能团的形成需要酶 PvdA，这是一种黄素依赖性单加氧酶，可催化 L-鸟氨酸的 N-5 羟基化。来自铜绿假单胞菌的pvdA在大肠杆菌中成功过表达，并首次纯化了该酶。该酶在pH 8.0时具有最大活性。在没有 L-鸟氨酸的情况下，PvdA 的 NADPH 氧化酶活性为 0.24 +/- 0.02 mu mol min(-1) mg(-1)。底物 L-鸟氨酸将这种活性刺激了 5 倍，并且该反应与羟胺的形成紧密耦合。该酶对 NADPH 和 Havin 腺嘌呤二核苷酸 (FAD(+)) 作为辅因子具有特异性，因为它不能利用 NADH 和 Havin 单核苷酸。通过荧光滴定，NADPH和FAD(+)的解离常数分别测定为105.6+/-6.0μM和9.9+/-0.3μM。稳态动力学分析表明，L-鸟氨酸依赖性NADPH氧化遵循Michaelis-Menten动力学，表观K-m和V-max值为0.58 mM和1.34 mu mol min(-1) mg(-1)。 L-赖氨酸是刺激NADPH氧化的非底物效应物，但发生解偶联并产生过氧化氢而不是羟基化L-赖氨酸。 L-2,4-二氨基丁酸、L-高丝氨酸和5-氨基戊酸不是底物或效应物，但它们是L-鸟氨酸依赖性NADPH氧化反应的竞争性抑制剂，K(ic)s为3至8 mM。结果表明效应子的化学性质对于模拟 PvdA 中 NADPH 氧化速率很重要。
Pseudonionas aeruginosa is an opportunistic pathogen that produces the siderophore pyoverdine, which enables it to acquire the essential nutrient iron from its host. Formation of the iron-chelating hydroxamate functional group in pyoverdine requires the enzyme PvdA, a flavin-dependent monooxygenase that catalyzes the N-5 hydroxylation of L-ornithine. pvdA from P. aeruginosa was successfully overexpressed in Escherichia coli, and the enzyme was purified for the first time. The enzyme possessed its maximum activity at pH 8.0. In the absence of L-ornithine, PvdA has an NADPH oxidase activity of 0.24 +/- 0.02 mu mol min(-1) mg(-1). The substrate L-ornithine stimulated this activity by a factor of 5, and the reaction was tightly coupled to the formation of hydroxylamine. The enzyme is specific for NADPH and Havin adenine dinucleotide (FAD(+)) as cofactors, as it cannot utilize NADH and Havin mononucleotide. By fluorescence titration, the dissociation constants for NADPH and FAD(+) were determined to be 105.6 +/- 6.0 mu M and 9.9 +/- 0.3 mu M, respectively. Steady-state kinetic analysis showed that the L-ornithine-dependent NADPH oxidation obeyed Michaelis-Menten kinetics with apparent K-m and V-max values of 0.58 mM and 1.34 mu mol min(-1) mg(-1). L-Lysine was a nonsubstrate effector that stimulated NADPH oxidation, but uncoupling occurred and hydrogen peroxide instead of hydroxylated L-lysine was produced. L-2,4-Diaminobutyrate, L-homoserine, and 5-aminopentanoic acid were not substrates or effectors, but they were competitive inhibitors of the L-ornithine-dependent NADPH oxidation reaction, with K(ic)s of 3 to 8 mM. The results indicate that the chemical nature of effectors is important for simulation of the NADPH oxidation rate in PvdA.