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首页> 外文期刊>Journal of Molecular Biology >Structural analysis and identification of PhuS as a heme-degrading enzyme from pseudomonas aeruginosa
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Structural analysis and identification of PhuS as a heme-degrading enzyme from pseudomonas aeruginosa

机译：铜绿假单胞菌血红素降解酶PhuS的结构分析与鉴定

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摘要

Bacterial pathogens require iron for proliferation and pathogenesis. Pseudomonas aeruginosa is a prevalent Gram-negative opportunistic human pathogen that takes advantage of immunocompromised hosts and encodes a number of proteins for uptake and utilization of iron. Here we report the crystal structures of PhuS, previously known as the cytoplasmic heme-trafficking protein from P. aeruginosa, in both the apo- and the holo-forms. In comparison to its homologue ChuS from Escherichia coli O157:H7, the heme orientation is rotated 180° across the α-γ axis, which may account for some of the unique functional properties of PhuS. In contrast to previous findings, heme binding does not result in an overall conformational change of PhuS. We employed spectroscopic analysis and CO measurement by gas chromatography to analyze heme degradation, demonstrating that PhuS is capable of degrading heme using ascorbic acid or cytochrome P450 reductase-NADPH as an electron donor and produces five times more CO than ChuS. Addition of catalase slows down but does not stop PhuS-catalyzed heme degradation. Through spectroscopic and mass spectrometry analysis, we identified the enzymatic product of heme degradation to be verdoheme. These data taken together suggest that PhuS is a potent heme-degrading enzyme, in addition to its proposed heme-trafficking function.
机译：细菌病原体需要铁才能增殖和发病。铜绿假单胞菌是一种普遍的革兰氏阴性机会性人类病原体，它利用免疫功能低下的宿主并编码许多蛋白质来吸收和利用铁。在这里，我们报告了载脂蛋白和完整形式的PhuS的晶体结构，以前称为铜绿假单胞菌的胞质血红素转运蛋白。与来自大肠杆菌O157：H7的同系物ChuS相比，血红素方向沿α-γ轴旋转180°，这可能解释了PhuS的某些独特功能特性。与以前的发现相反，血红素结合不会导致PhuS的整体构象变化。我们采用光谱分析和气相色谱法测定一氧化碳，以分析血红素的降解情况，表明PhuS能够使用抗坏血酸或细胞色素P450还原酶-NADPH作为电子供体来降解血红素，并且产生的二氧化碳比ChuS高五倍。过氧化氢酶的添加减慢了速度，但并未阻止PhuS催化的血红素降解。通过光谱和质谱分析，我们确定了血红素降解的酶促产物是verdoheme。这些数据加在一起表明，PhuS除了提议的血红素转运功能外，还是一种有效的血红素降解酶。

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