title: Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis.
abstract: AhpF, a homodimer of 57 kDa subunits, is a flavoenzyme which catalyzes the NADH-dependent reduction of redox-active disulfide bonds in the peroxidase AhpC, a member of the recently identified peroxiredoxin class of antioxidant enzymes. The structure of AhpF from Salmonella typhimurium at 2.0 A resolution, determined using multiwavelength anomalous dispersion, shows that the C-terminal portion of AhpF (residues 210-521) is structurally like Escherichia coli thioredoxin reductase. In addition, AhpF has an N-terminal domain (residues 1-196) formed from two contiguous thioredoxin folds, but containing just a single redox-active disulfide (Cys129-Cys132). A flexible linker (residues 197-209) connects the domains, consistent with experiments showing that the N-terminal domain acts as an appended substrate, first being reduced by the C-terminal portion of AhpF, and subsequently reducing AhpC. Modeling studies imply that an intrasubunit electron transfer accounts for the reduction of the N-terminal domain in dimeric AhpF. Furthermore, comparing the N-terminal domain with protein disulfide oxidoreductase from Pyrococcus furiosis, we describe a new class of protein disulfide oxidoreductases based on a novel mirror-image active site arrangement, with a distinct carboxylate (Glu86) being functionally equivalent to the key acid (Asp26) of E. coli thioredoxin. A final fortuitous result is that the N-terminal redox center is reduced and provides a high-resolution view of the thiol-thiolate hydrogen bond that has been predicted to stabilize the attacking thiolate in thioredoxin-like proteins.
authors: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.
authors: Z A
authors: ZA
authors: Wood
authors: L B
authors: LB
authors: Poole
authors: P A
authors: PA
authors: Karplus
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D054464:Peroxiredoxins; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D054481:Thioredoxin Reductase 1; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D013879:Thioredoxins; D010544:Peroxidases; D054464:Peroxiredoxins; C515937:ahpC protein, E coli; C515938:ahpF protein, E coli; D054481:Thioredoxin Reductase 1; D013880:Thioredoxin-Disulfide Reductase