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首页> 美国卫生研究院文献>International Journal of Molecular Sciences >Identification of Active Site Residues of the Siderophore Synthesis Enzyme PvdF and Evidence for Interaction of PvdF with a Substrate-Providing Enzyme
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Identification of Active Site Residues of the Siderophore Synthesis Enzyme PvdF and Evidence for Interaction of PvdF with a Substrate-Providing Enzyme

机译：鉴定Sircleophore合成酶PVDF的活性位点残留物和PVDF与基材提供酶的依据的证据

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摘要

The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host–pathogen interactions.
机译：有问题的机会理性病原体假单胞菌铜绿假单胞菌分泌了一个冰川的冰川。百伏的细菌可释放熨斗，用于生长和致病性在一系列不同的感染模型中。 PVDF是一种羟基呋喃胺转化酶酶，对于百voRdine合成，催化合成的葡萄酒（Fohorn）的催化合成，其形成百倍分子的一部分，并提供铁螯合的羟肟酸酯配体。使用质谱法测定，我们确认纯化的PVDF催化来自羟（甲醛丁酸亚甲酸盐底物的FoHorn。进行现场定向诱变，以研究预测酶活性所需的氨基酸残基。通过测量其将灰叶产生至PVDF突变菌株的能力，在体外和体内测定酶变体的活性。两个推定的催化残基N168和H170的变体大大降低了体内酶活性，但在体外没有废除活性。在体内和体外改变第三种残留物D229废除活性。预测阻断N10-甲酰基四氢脱氢（Fthf）进入活性位点的变化，也在体外和体内废除活性。共纯化测定表明，PVDF与催化羟基植物合成的酶PVDA结合，并且该相互作用可能提高FOHORN合成的效率。我们的研究结果推进了铜绿假单胞菌如何合成百罗氏素，其宿主病原体相互作用的关键因素。

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