| The alkyl hydroperoxide reductase (AhpR) system plays an essential role in reducing a variety of toxic hydroperoxide substrates in many eubacteria. AhpR serves to protect the cell against oxidative stress for staying bacterial fitness. The alkyl hydroperoxide reductase includes two subunits, AhpF and AhpC. AhpC is a member of typical 2-Cys peroxiredoxin, which directly respond to converting hydroperoxide substrates to water or the corresponding alcohols for detoxification. AhpF, a flavin-protein, regenerates AhpC by transferring electrons from NADH onto AhpC. AhpF consists of three catalytic centers for transferring electrons from initial NADH onto the final AhpC:an N-terminal redox-active disulfide-containing domain (NTD), an FAD-bound domain (FAD domain), and an NADH binding redox-active disulfide-containing domain (NAD domain). The catalytic process mediated by AhpF is believed to follow a path similar to that performed by the thioredoxin reductase and thioredoxin system (TrxR-Trx). First, the electron transfer intramolecularly flows in AhpF, from pyridine nucleotide binding in the NAD domain to the flavin in the FAD domain, then back to the second catalytic site of Cys345-Cys348 redox-active center in the NAD domain and subsequent the Cysl29-Cys132 center in NTD. Finally, the electron intermolecularly transfers from Cys129-Cys132 center in NTD of AhpF to the dimeric AhpC Cys47-Cys166’center. During the process, Cys348 of AhpF as the nucleophile attacks the Cys129 sulfur in a disulfide bond form, then the thiolate of Cys129 attacks Cys166 of the intersbuunit disfulfide bond within an AhpC dimer. All these steps necessitate AhpF to undergo very large conformational changes to allow for Cys129-Cysl32 to communicate with the Cys345-Cys348 to receive the NADH-derived electron, then rearrange away from this position for channeling electron to its cognate substrate AhpC.We purified the endogenous AhpF protein from E.coli and determined its crystal structure at a resolution of 2.2 A. Overall, the AhpF structure reveals a novel open conformation with its N-terminal domain far away from its C-terminal domains, different from that of its highly homologous StAhpF from Salmonella typhimurium (StAhpF) and a recent solved E.coli AhpF structure (EcAhpF). This structure provides evidence that AhpF might utilize an open state to catalyze AhpC decamer more efficiently. The open conformation of AhpF we observe in the crystal structure might represent an intermediate state for AhpF ready for the electron intermolecular transfer.Peptidoglycan (PG, or murein) is an essential and unique component of the bacterial cell wall outside the cytoplasmic membrane, whose main functions are to preserve cell integrity by withstanding the high turgor pressure and to maintain a defined cell shape. PgrR is a transcription factor functioning as a repressor of the expression of genes involved in PG degradation. In detail, it directly negatively regulates the expression of the ycjY-ymjD-ymjC-mpaA operon by binding to the PgrR-box. The genetic coupling of these genes and their related biochemical functions suggest that YcjY may be a novel hydrolase participated in the PG degradation. Recently it is reported that the over-expression of cloned ycjY induces an intriguing phenomenon:filamentous phenotype and YcjY may play an as yet unidentified role in E.coli cell division. In fact, PG hydrolases are essential for cell shape determination. Here, we report the crystal structure of E. coli uncharacterized protein YcjY at 2.0 A. The structure of YcjY monomer presents a canonical α/β hydro lase fold, which can be split into two parts:a catalytic core domain and an insertion (Ilel44-Tyr240). The core α/β hydro lase domain has a central eight stranded mainly parallel P-sheet with a single antiparallel strand β2. The central sheet is sandwiched on each side by a helices, three helices (α1, α11, and α12) are located on one side of the sheet and three helices (α2, α3, and α10) are located on the other. The central β-sheet shows the typical left handed superhelical twist with strands β1 and β10 crossing each other at an angle of approximately 90℃. The insertion is located in the loop between β6 and β9 at the C-terminal end of the central β-sheet. The insertion consists of one small antiparallel β-hairpin (β7/β8) and six a-helices (α4, α5, α6, α7, α8 and α9), all of which forms a lid domain above the catalytic core domain. The oligomerization status of YcjY in solution was investigated by using gel filtration chromatography, which showed a peak with apparent molecular weight of 58.88 kDa, strongly suggestive of a dimer in solution (monomer Mr=33.7 kDa). Compared with other α/β hydrolases, we found its active sites and identified these sites in vivo through filamentous phenotype. In addition, the function of YcjY may be influenced by the co-expression of ymjD, ymjC and mpaA in the same operon. Taken together, YcjY may be a novel α/β hydrolase that participate in the PG degradation. |
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