Oxidative folding and reductive activities of EhPDI, a protein disulfide isomerase from Entamoeba histolytica.
PDI enzymes are oxidoreductases that catalyze oxidation, reduction and isomerization of disulfide bonds in polypeptide substrates. We have previously identified an E. histolytica PDI enzyme (EhPDI) that exhibits oxidase activity in vivo. However, little is known about the specific role of its redox-related structural features on the enzymatic activity. Here, we have studied the in vivo oxidative folding of EhPDI by mutagenic analysis and functional complementation assays as well as the in vitro oxidative folding and reductive activities by comparative kinetics using functional homologues in standard assays. We have found that the active-site cysteine residues of the functional domains (Trx-domains) are essential for catalysis of disulfide bond formation in polypeptides and proteins, such as the bacterial alkaline phosphatase. Furthermore, we have shown that the recombinant EhPDI enzyme has some typical properties of PDI enzymes: oxidase and reductase activities. These activities were comparable to those observed for other functional equivalents, such as bovine PDI or bacterial thioredoxin, under the same experimental conditions. These findings will be helpful for further studies intended to understand the physiological role of EhPDI.
Facultad de Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja California, Calzada Tecnológico 14418, Tijuana, Baja California, 22390, México.
Rosa E
RE
Mares
Paloma D
PD
Magaña
Samuel G
SG
Meléndez-López
Alexei F
AF
Licea
José M
JM
Cornejo-Bravo
Marco A
MA
Ramos
D000818:Animals; D020134:Catalytic Domain; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D009154:Mutation; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases