Replacement of Pro109 by His in TlpA, a thioredoxin-like protein from Bradyrhizobium japonicum, alters its redox properties but not its in vivo functions.
TlpA, the membrane-anchored, thioredoxin-like protein from Bradyrhizobium japonicum, is essential for cytochrome aa3 biogenesis. The periplasmic domain of TlpA was previously shown to have protein thiol:disulfide oxidoreductase activity and reducing properties similar to those of cytoplasmic thioredoxins. Here, we replaced the proline-109 in its active-site sequence C107 V108 P109 C110 by a histidine residue. The resulting active-site motif (CVHC) resembles that of oxidizing thiol:disulfide oxidoreductases such as protein disulfide isomerase (PDI) and DsbA. Indeed, the TlpA variant P109H was by 66 mV more oxidizing than the wild-type protein. Nevertheless, the altered protein was even more efficient in catalyzing the reduction of insulin disulfides by dithiothreitol than the wild-type due to a faster recycling of its catalytically active, reduced form. Cells of B. japonicum expressing only the mutated tlpA gene had the same phenotypes as wild-type cells, suggesting that the change in the redox potential of TlpA was not critical for its in vivo function.
Rossmann
R
R
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Stern
D
D
Loferer
H
H
Jacobi
A
A
Glockshuber
R
R
Hennecke
H
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004229:Dithiothreitol / Q000378:metabolism; D005798:Genes, Bacterial; D005978:Glutathione / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007328:Insulin / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011392:Proline / Q000737:chemistry; D011490:Protein Disulfide Reductase (Glutathione) / Q000378:metabolism; D012230:Rhizobiaceae / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D007328:Insulin; C084726:TlpA protein, Bradyrhizobium; D006639:Histidine; D013879:Thioredoxins; D011392:Proline; D011490:Protein Disulfide Reductase (Glutathione); D005978:Glutathione; D004229:Dithiothreitol