Oxidative protein folding in the mitochondrial intermembrane space requires the transfer of a disulfide bond from MIA40 to the substrate. During this process MIA40 is reduced and regenerated to a functional state through the interaction with the flavin-dependent sulfhydryl oxidase ALR. Here we present the mechanistic basis of ALR-MIA40 interaction at atomic resolution by biochemical and structural analyses of the mitochondrial ALR isoform and its covalent mixed disulfide intermediate with MIA40. This ALR isoform contains a folded FAD-binding domain at the C-terminus and an unstructured, flexible N-terminal domain, weakly and transiently interacting one with the other. A specific region of the N-terminal domain guides the interaction with the MIA40 substrate binding cleft (mimicking the interaction of the substrate itself), without being involved in the import of ALR. The hydrophobicity-driven binding of this region ensures precise protein-protein recognition needed for an efficient electron transfer process.

The gene encoding alkaline phosphatase (AP) from the psychrophilic strain TAB5 was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1125 base pairs which encodes a polypeptide consisting of signal peptide of 22 amino acids and a mature protein of 353 amino acids was identified. The deduced protein sequence of AP exhibits a 38% identity to the AP III and AP IV sequences of Bacillus subtilis and conserves the typical sequence motifs of the core structure and active sites of APs from various sources. Based on the crystal structure of the mutated Escerichia coli AP D153H, a homology-based 3D model of the TAB5 AP was constructed on the basis of which various features of the enzyme amino-acid sequence can be interpreted in terms of potential psychrophilic adaptations.The AP gene was expressed in E. coli BL21(DE3) cells, the recombinant protein was isolated to homogeneity from the membrane fraction of the cells and its properties were examined. The purified TAB5 AP shows typical features of a cold enzyme: high catalytic activity at low temperature and a remarkable thermosensitivity.The use of this heat-labile enzyme, for dephosphorylation of nucleic acids, simplifies dephosphorylation protocols.Keywords: alkaline phosphatase; homology modelling; psychrophilic enzyme; purification.Alkaline phosphatases (APs) are dimeric, zinc-containing nonspecific phosphomonoesterases that exist in various organisms from bacteria to mammals [1]. Sequence comparisons of APs from a variety of species combined with structural information from the Escherichia coli AP [2±4], suggest that the functionally important domains are conserved [5±7]. However, although the amino-acid sequences of AP from bacteria and mammals show sequence conservation of 25±30%, mammalian APs are 10±20 times more active than the E. coli enzyme [8].Comparisons between the E. coli and the mammalian enzymes reveal two striking differences close to the active site: in the E. coli AP, two of the catalytic residues (153 and 328) are Asp and Lys, while in the mammalian enzymes the equivalent residues are both His. Interestingly, the Asp3His substitution at position 153 in the E. coli AP results in a mutant enzyme (D153H), which exhibits typical properties of a mammalian AP, including a shift in the pH of optimal activity, low activity in the absence of Mg 2+ ions, a time dependent activation by Mg 2+ ions and a reduction in activity in the presence of Zn 2 ions [9,10].Although, the origin of the observed properties of the D153H mutant is not yet well understood, the molecular basis of increased enzymatic activity represents a problem which is both challenging and biotechnologically important. Generally, higher catalytic efficiencies at low and moderate temperatures, along with remarkable thermolability are properties of psychrophilic enzymes [11,12]. Therefore, an AP from psychrophiles exhibiting such properties could be of significant biotechnological interest, while on the other hand it could provide an attractive model system...

Lionaki, E., Aivaliotis, M., Pozidis, C., and Tokatlidis, K. (2010) The Nterminal shuttle domain of Erv1 determines the affinity for Mia40 and mediates electron transfer to the catalytic Erv1 core in yeast mitochondria. Antioxidants and Redox Signaling, 13 (9 AbstractErv1 and Mia40 constitute the two important components of the disulfide relay system that mediates oxidative protein folding in the mitochondrial intermembrane space. Mia40 is the import receptor that recognizes the substrates introducing disulfide bonds while it is reduced. A key function of Erv1 is to recycle Mia40 to its active oxidative state. Our aims here were to dissect the domain of Erv1 that mediates the protein-protein interaction with Mia40 and to investigate the interactions between the shuttle domain of Erv1 and its catalytic core and their relevance for the interaction with Mia40. We purified these domains separately as well as cysteine mutants in the shuttle and the active core domains. The noncovalent interaction of Mia40 with Erv1 was measured by isothermal titration calorimetry, whereas their covalent mixed disulfide intermediate was analyzed in reconstitution experiments in vitro and in organello. We established that the N-terminal shuttle domain of Erv1 is necessary and sufficient for interaction to occur. Furthermore, we provide direct evidence for the intramolecular electron transfer from the shuttle cysteine pair of Erv1 to the core domain. Finally, we reconstituted the system by adding in trans the N-and C-terminal domains of Erv1 together with its substrate Mia40. Antioxid. Redox Signal. 13, 1327-1339.