title: Atypical protein disulfide isomerases (PDI): Comparison of the molecular and catalytic properties of poplar PDI-A and PDI-M with PDI-L1A.
abstract: 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.
authors.affiliation: UMR 1136 Interactions Arbres/Microorganismes, Université de Lorraine/ INRA, Faculté des Sciences et Technologies, Vandoeuvre-lès-Nancy, France.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Selles
authors.affiliation: UMR 1136 Interactions Arbres/Microorganismes, Université de Lorraine/ INRA, Faculté des Sciences et Technologies, Vandoeuvre-lès-Nancy, France.
authors.forename: Flavien
authors.initials: F
authors.lastname: Zannini
authors.affiliation: UMR 1136 Interactions Arbres/Microorganismes, Université de Lorraine/ INRA, Faculté des Sciences et Technologies, Vandoeuvre-lès-Nancy, France.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Couturier
authors.affiliation: UMR 1136 Interactions Arbres/Microorganismes, Université de Lorraine/ INRA, Faculté des Sciences et Technologies, Vandoeuvre-lès-Nancy, France.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Jacquot
authors.affiliation: UMR 1136 Interactions Arbres/Microorganismes, Université de Lorraine/ INRA, Faculté des Sciences et Technologies, Vandoeuvre-lès-Nancy, France.
authors.forename: Nicolas
authors.identifier: http://orcid.org/0000-0002-2036-7884
authors.initials: N
authors.lastname: Rouhier
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D050538:Malate Dehydrogenase (NADP+) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054464:Peroxiredoxins / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013440:Sulfides / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D007328:Insulin; D020033:Protein Isoforms; D013440:Sulfides; D004228:Dithionitrobenzoic Acid; D007501:Iron; D010088:Oxidoreductases; D050538:Malate Dehydrogenase (NADP+); D054464:Peroxiredoxins; D013880:Thioredoxin-Disulfide Reductase; D019704:Protein Disulfide-Isomerases