Atypical protein disulfide isomerases (PDI): Comparison of the molecular and catalytic properties of poplar PDI-A and PDI-M with PDI-L1A

• Published in: PloS one 2017-03, Vol.12 (3), p.e0174753-e0174753
• Main Authors: Selles, Benjamin, Zannini, Flavien, Couturier, Jérémy, Jacquot, Jean-Pierre, Rouhier, Nicolas
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Biology and life sciences; Catalysis; Catalysts; Chemical bonds; Chemical reactions; Chemical reduction; Chromatography; Disulfide bonds; Dithionitrobenzoic Acid - metabolism; E coli; Endoplasmic reticulum; Enzymes; Health aspects; Humans; Hydrogen-Ion Concentration; Insulin; Insulin - metabolism; Iron - metabolism; Isoforms; Isomerization; Life Sciences; Malate; Malate dehydrogenase; Malate Dehydrogenase (NADP+) - chemistry; Malate Dehydrogenase (NADP+) - genetics; Malate Dehydrogenase (NADP+) - metabolism; Medicine and Health Sciences; Modules; Mutagenesis; Mutagenesis, Site-Directed; NADP; Nicotinamide adenine dinucleotide phosphate; Oxidation; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism; Peroxiredoxins - metabolism; pH effects; Physical Sciences; Physiology; Protein disulfide isomerases; Protein Disulfide-Isomerases - chemistry; Protein Disulfide-Isomerases - genetics; Protein Disulfide-Isomerases - metabolism; Protein folding; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Proteins; Recombinant proteins; Redox properties; Reductase; Reductases; Research and analysis methods; Ribonuclease A; Sequence Homology, Amino Acid; Substrates; Sulfides - metabolism; Thioredoxin; Thioredoxin-Disulfide Reductase - chemistry; Thioredoxin-Disulfide Reductase - genetics; Thioredoxin-Disulfide Reductase - metabolism; Thioredoxins
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0174753

Protein disulfide isomerases are overwhelmingly multi-modular redox catalysts able to perform the formation, reduction or isomerisation of disulfide bonds. We present here the biochemical characterization of three different poplar PDI isoforms. PDI-A is characterized by a single catalytic Trx module, the so-called a domain, whereas PDI-L1a and PDI-M display an a-b-b'-a' and a°-a-b organisation respectively. Their activities have been tested in vitro using purified recombinant proteins and a series of model substrates as insulin, NADPH thioredoxin reductase, NADP malate dehydrogenase (NADP-MDH), peroxiredoxins or RNase A. We demonstrated that PDI-A exhibited none of the usually reported activities, although the cysteines of the WCKHC active site signature are able to form a disulfide with a redox midpoint potential of -170 mV at pH 7.0. The fact that it is able to bind a [Fe2S2] cluster upon Escherichia coli expression and anaerobic purification might indicate that it does not have a function in dithiol-disulfide exchange reactions. The two other proteins were able to catalyze oxidation or reduction reactions, PDI-L1a being more efficient in most cases, except that it was unable to activate the non-physiological substrate NADP-MDH, in contrast to PDI-M. To further evaluate the contribution of the catalytic domains of PDI-M, the dicysteinic motifs have been independently mutated in each a domain. The results indicated that the two a domains seem interconnected and that the a° module preferentially catalyzed oxidation reactions whereas the a module catalyzed reduction reactions, in line with the respective redox potentials of -170 mV and -190 mV at pH 7.0. Overall, these in vitro results illustrate that the number and position of a and b domains influence the redox properties and substrate recognition (both electron donors and acceptors) of PDI which contributes to understand why this protein family expanded along evolution.