ABSTRACT
The ability of the vancomycin-resistant Enterococcus faecalis (V583) to restore redox homeostasis via antioxidant defense mechanism is of importance, and knowledge into this defense is essential to understand its antibiotic-resistance and survival in hosts. The flavoprotein disulfide reductase AhpR, composed of the subunits AhpC and AhpF, represents one such vital part. Circular permutation was found to be a feature of the AhpF protein family. E. faecalis (V583) AhpF (EfAhpF) appears to be a representative of a minor subclass of this family, the typically N-terminal two-fold thioredoxin-like domain (NTD_N/C) is located at the C-terminus, whereas the pyridine nucleotide-disulfide oxidoreductase domain is encoded in the N-terminal part of its sequence. In EfAhpF, these two domains are connected via an unusually long linker region providing optimal communication between both domains. EfAhpF forms a dimer in solution similar to Escherichia coli AhpF. The crystallographic 2.3Å resolution structure of the NTD_N/C domain reveals a unique loop-helix stretch (409ILKDTEPAKELLYGIEKM426) not present in homologue domains of other prokaryotic AhpFs. Deletion of the unique 415PAKELLY421-helix or of 415PAKELL420 affects protein stability or attenuates peroxidase activity. Furthermore, mutation of Y421 is described to be essential for E. faecalis AhpF's optimal NADH-oxidative activity.
