Attachment of the N-terminal domain of Salmonella typhimurium AhpF to Escherichia coli thioredoxin reductase confers AhpC reductase activity but doesnot affect thioredoxin reductase activity

AhpF of Salmonella typhimurium, the flavoprotein reductase required for cat alytic turnover of AhpC with hydroperoxide substrates in the alkyl hydroper oxide reductase system, is a 57 kDa protein with homology to thioredoxin re ductase (TrR) from Escherichia coli. Like TrR, AhpF employs tightly bound F AD and redox-active disulfide center(s) in catalyzing electron transfer fro m reduced pyridine nucleotides to the disulfide bond of its protein substra te, Homology of AhpF to the smaller (35 kDa) TrR protein occurs in the C-te rminal part of AhpF; a stretch of about 200 amino acids at the N-terminus o f AhpF; contains an additional redox-active disulfide center and is require d for catalysis of AhpC reduction. We have demonstrated that fusion of the N-terminal 207 amino acids of AhpF to full-length TrR results in a chimeric protein (Nt-TrR) with essentially the same catalytic efficiency (k(cat)/K- m) as AhpF in AhpC reductase assays; both k(cat) and the K-m for AhpC are d ecreased about 3-4-fold for Nt-TrR compared with AhpF. in addition, Nt-TrR retains essentially full TrR activity. Based on results from two mutants of Nt-TrR (C129,132S and C342,345S), AhpC reductase activity requires both ce nters while TrR activity requires only the C-terminal-most disulfide center in Nt-TrR. The high catalytic efficiency with which Nt-TrR can reduce thio redoxin implies that the attached N-terminal domain does not block access o f thioredoxin to the TrR-derived Cys342-Cys345 center of Nt-TrR nor does it impede the putative conformational changes that this part of Nt-TrR is pro posed to undergo during catalysis. These studies indicate that the C-termin al part of AhpF and bacterial TrR have very similar mechanistic properties. These findings also confirm that the N-terminal domain of AhpF plays a dir ect role in AhpC reduction.