Biochemical characterization of a flavin adenine dinculeotide-dependent monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism
Biochemical characterization of a flavin adenine dinculeotide-dependent monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism
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DOI：
10.1021/bi700932q
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发表时间：
2007-10-23
期刊：
影响因子：
2.9
通讯作者：
Lamb, Audrey L.
中科院分区：
文献类型：
--
作者：
Meneely, Kathleen M.;Lamb, Audrey L.
吡咯菌素是条件致病菌铜绿假单胞菌在人类宿主铁限制条件下产生的羟甲酸铁载体。这个铁载体包括作为铁络合剂的多肽骨架中的鸟氨酸的衍生物。PvdA是鸟氨酸羟化酶，在制备这些衍生物的过程中执行第一步酶促反应。PvdA需要黄素腺嘌呤二核苷酸(FAD)和烟酰胺腺嘌呤二核苷酸磷酸(NADPH)才能发挥活性；它被发现是一种可溶性单体，在pH值为8.0时最具活性。该酶在NADPH氧化实验中表现为Michaelis-Menten动力学，但在羟化实验中显示在较高的鸟氨酸浓度下底物抑制。PvdA对底物和辅酶都具有高度的特异性，赖氨酸被证明是该酶相对于鸟氨酸的非底物效应物和混合抑制物。氯化物是PvdA相对于鸟氨酸的混合抑制剂，但对于NADPH是竞争性抑制剂，而大块汞化合物(对氯汞苯甲酸酯)是相对于鸟氨酸的混合抑制剂。稳态实验表明，PvdA/FAD与NADPH和鸟氨酸形成三元络合物用于催化。在没有鸟氨酸的情况下，PvdA表现出NADPH对黄素的缓慢底物非依赖性还原。将PvdA与荧光假单胞菌的对羟基苯甲酸羟基酶(PHBH)和含有黄素的单加氧酶(来自裂殖酵母和猪肝微粒体的FMOS)进行生化比较，得出PvdA催化通过一种新的反应机制进行的假设。
Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) for activity; it was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics in an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a nonsubstrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA with respect to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (p-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady-state experiments indicate that PvdA/FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to p-hydroxybenzoate hydroxylase (PHBH, from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs, from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism.