Spectroscopic Determination of Distinct Heme Ligands in Outer-Membrane Receptors PhuR and HasR of Pseudomonas aeruginosa.
Spectroscopic Determination of Distinct Heme Ligands in Outer-Membrane Receptors PhuR and HasR of Pseudomonas aeruginosa.
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DOI：
10.1021/acs.biochem.5b00017
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发表时间：
2015-04-28
期刊：
影响因子：
2.9
通讯作者：
Wilks A
中科院分区：
文献类型：
--
作者：
Smith AD;Modi AR;Sun S;Dawson JH;Wilks A
铜绿假单胞菌PAO1编码两个外膜受体，PhuR（假单胞菌血红素摄取）和HasR（血红素同化系统）。HasR受体通过与分泌的血红蛋白HasAp相互作用获得血红素。非血细胞依赖性PhuR与血红素转运到细胞质所需的蛋白质一起编码。在此，我们报道了HasR和PhuR受体的分离和表征。吸收和MCD光谱证实，与其他革兰氏阴性OM受体类似，HasR通过表面暴露的frap环的保守n端塞His-221和His-624来定位血红素。相比之下，PhuR表现出明显的吸收和MCD光谱，与Tyr残基的配位一致。PhuR与所有已知革兰氏阴性OM血红素受体的序列比对显示，在FRAP环中缺乏保守的His，但在519和529位置缺少两个Tyr残基。位点定向突变和光谱鉴定证实了tyrr -519和n端插入His-124提供了PhuR中的血红素配体。我们认为PhuR和HasR代表了能够在宿主定植和感染的广泛生理条件下感知和获取血红素的非冗余血红素受体。
Pseudomonas aeruginosa PAO1 encodes two outer membrane receptors, PhuR (Pseudomonas heme uptake) and HasR (heme assimilation system). The HasR receptor acquires heme through interaction with a secreted hemophore, HasAp. The non-hemophore-dependent PhuR is encoded along with proteins required for heme translocation into the cytoplasm. Herein, we report the isolation and characterization of the HasR and PhuR receptors. Absorption and MCD spectroscopy confirmed that, similar to other Gram-negative OM receptors, HasR coordinates heme through the conserved N-terminal plug His-221 and His-624 of the surface-exposed FRAP-loop. In contrast, PhuR showed distinct absorption and MCD spectra consistent with coordination through a Tyr residue. Sequence alignment of PhuR with all known Gram-negative OM heme receptors revealed a lack of a conserved His within the FRAP loop but two Tyr residues at positions 519 and 529. Site-directed mutagenesis and spectroscopic characterization confirmed Tyr-519 and the N-terminal plug His-124 provide the heme ligands in PhuR. We propose that PhuR and HasR represent nonredundant heme receptors capable of sensing and accessing heme across a wide range of physiological conditions on colonization and infection of the host.