Bacillus licheniformis 유래 PerR 유사 단백질들의 과산화물 감지 분자 메커니즘 연구

Abstract

It is now appreciated that there are diverse subtypes of Fur family proteins, Fur, Zur, Mur, and Nur, which are specialized to sense iron, zinc, manganese, and nickel, respectively. In addition, the Fur family also includes sensors of heme (Irr) and peroxide (PerR). A model Gram-positive bacterium, Bacillus subtilis contains three Fur family regulators including Fur, Zur and PerR. However, here I identified that B. licheniformis, a close relative of B. subtilis, encodes five Fur family proteins: two novel PerR-like proteins (PerR2 and PerR3) in addition to Fur (FurBL), Zur (ZurBL), and PerR (PerRBL) orthologues. Each of the five proteins contains a stable structural Zn2+ binding site composed of four conserved cysteine residues. Mass spectrometry data indicate that the two PerR-like proteins (PerR2 and PerR3) also can sense H2O2 by histidine oxidation but with different sensitivity when compared to that of PerRBL. These results suggest that B. licheniformis engages three PerR subfamily proteins as peroxide sensors. Here I investigated a detailed H2O2 sensing mechanism of PerRBL as a major peroxide sensing regulator, and its physiological role in B. licheniformis. As noted for PerRBS, PerRBL responds to H2O2 by Fe2+-dependent protein oxidation and regulates the expression of same set of defense genes known as PerR regulon in B.subtilis. Among these, four (katA, mrgA, ahpCF, and zosA) genes were induced under H2O2-stressed conditions, whereas others (hemA, fur, and perR) did not respond to H2O2. Interestingly, it was revealed that expression of PerRBL was essentially required for the growth of B. licheniformis. Further genetic studies demonstrated that the lack of growth of the perR mutant strain was due to the starvation of intracellular iron, and could be restored by additional mutation of fur. These results suggest that PerR-mediated regulation of fur is important for maintaining suitable iron levels inside cell. One of PerR paralogues, PerR2 was characterized as a secondary H2O2 sensor in B. licheniformis since PerR2 exhibited both PerR-like repressor activity and H2O2 responsiveness in vivo. Here I provide several evidences that the apparent weak repressor activity of PerR2 is due to the hypersensitivity of Fe2+-bound PerR2 under aerobic condition. PerR2 expressed in cells grown under anaerobic or iron-depleted condition, existed as nearly reduced status, and exhibited increased repressor activity. Additionally, I identified the key amino acid residues which control the intrinsic H2O2 sensitivity of PerR2. A substitution of Asp86 or Asp105 residue with Glu rendered PerR2 more resistant to oxidation by H2O. All these data support the idea that PerR2 senses extremely low levels of H2O2 which cannot be normally sensed by major H2O2 sensor PerRBL. Finally, further genetic and biochemical studies revealed that PerR2 is specifically induced and can function like PerRBL under zinc-depleted conditions. PerR3 was found as not a canonical PerR-like protein since it existed as a monomer lacking DNA-binding activity. However, PerR3, as a member of PerRBL regulon, was massively induced under H2O2-stressed condition. Moreover, cells producing PerR3 showed increased H2O2 resistance with the induction of other PerRBL-regulated genes, indicating that the role of PerR3 is involved in PerRBL-mediated H2O2 sensing mechanism. Here I propose the novel H2O2 sensing mechanism, where PerR3 acts as an anti-repressor of PerRBL. PerR3 forms heterodimer specifically with PerRBL, as judged by several protein-protein interaction analyses. Furthermore, repressor activity of single-chained heterodimer was relieved when compared with that of PerRBL homodimer. Surprisingly, mutant PerR3 protein lacking regulatory metal binding exhibited far more strengthened anti-repressor activity than wild type PerR3, suggesting that regulatory metal binding in PerR3 is important factor for the control of anti-repressor activity of PerR3.