Leishmania parasites primarily infect cells of macrophage lineage and can cause leishmaniasis in the skin, mucosal, and visceral organs, depending on both host-and parasite-derived factors. The protein disulfide isomerases (PDIs) are thiol-disulfide oxidoreductases that catalyze the formation, reduction, and isomerization of disulfide bonds of proteins in cells. Although four Leishmania PDI genes are functionally inferred from homology in the genome sequences, only two of them have been expressed as active proteins to date. The functional relationship among various PDI enzymes remains largely unclear. In this study, we expressed and partially characterized all four L. amazonensis PDIs encoding 52-, 47-, 40-, and 15-kDa proteins. Homology analysis showed that the sequence identity between L. amazonensis (New World) PDIs and their counterpart PDI sequences from L. major (Old World) ranged from 76% to 99%. Kinetic characterization indicated that while the 15-, 40-, and 47-kDa PDI proteins displayed both insulin isomerase and reductase activities, the 52-kDa protein had only isomerase activity with no detectable reductase activity. All four PDI proteins were recognized by sera from L. amazonensis-infected mice and were sensitive to inhibition by standard PDI inhibitors. This study describes the enzymatic activities of recombinant L. amazonensis PDIs and suggests a role for these proteins in parasite development.

To avoid nonspecific and/or undesirable binding and reactivity of metal ions with cellular components, organisms have evolved metal-specific systems for trafficking proteins. Although systems differ, those handling soft metal ions such as Hg(2+), Cu(+), Zn(2+), etc., all utilize heavy metal-associated (HMA) proteins and domains of ~70 amino acids with a conserved GMXCXXC motif in a βαββαβ structural fold. While the conserved cysteines define a common metal binding site in these proteins, other structural features must be utilized to create metal ion, protein partner, and contextual specificities. This paper presents initial structure-function studies of the N-terminal HMA domain (NmerA) of Tn501 mercuric ion reductase (MerA) aimed at identifying structural features critical to its role in facilitating efficient transfer of Hg(2+) to the MerA catalytic core for reductive detoxification. First, NMR solution structures of reduced and Hg(2+)-bound forms of NmerA are presented that allow definition and comparison of the structure of the metal binding loop in the two states. Structural differences between the two forms are compared with differences observed in three HMA domains with different metal ion and functional contexts. Second, analyses of the UV absorbance properties of wild-type, Cys11Ala, and Cys14Ala forms of NmerA are presented that provide assignments of the pK(a) values for the two cysteine thiols of the metal binding motif. Third, results from ¹³C NMR studies with wild-type and Y62F NmerA labeled with [β-¹³C]cysteine are presented that define a role for Tyr62 in modulating the pK(a) values of the cysteine thiols.

Aerobic and facultative bacteria and archaea harboring mer loci exhibit resistance to the toxic effects of Hg(II) and organomercurials [RHg(I)]. In broad spectrum resistance, RHg(I) is converted to less toxic Hg(0) in the cytosol by the sequential action of organomercurial lyase (MerB: RHg(I) --> RH + Hg(II)) and mercuric ion reductase (MerA: Hg(II) --> Hg(0)) enzymes, requiring transfer of Hg(II) from MerB to MerA. Although previous studies with γ-proteobacterial versions of MerA and a non-physiological Hg(II)-DTT-MerB complex qualitatively support a pathway for direct transfer between proteins, assessment of the relative efficiencies of Hg(II) transfer to the two different di-cysteine motifs in γ-proteobacterial MerA and to competing cellular thiol is lacking. Here we show the intrinsic tryptophan fluorescence of γ-proteobacterial MerB is sensitive to Hg(II) binding and use this to probe the kinetics of Hg(II) removal from MerB by the N-terminal domain (NmerA) and catalytic core C-terminal cysteine pairs of its co-evolved MerA, and by glutathione (GSH), the major competing cellular thiol in γ-proteobacteria. At physiologically relevant concentrations, reaction with a 10-fold excess NmerA over HgMerB removes ≥ 92% Hg(II), while similar extents of reaction require more than 1000-fold excess of GSH. Kinetically, the apparent second order rate constant for Hg(II) transfer from MerB to NmerA, at 2.3 ± 0.1 × 104 M−1 s−1 is ~ 100-fold greater than that for GSH (1.2 ± 0.2 × 102 M−1 s−1) or the MerA catalytic core (1.2 × 102 M−1 s−1), establishing transfer to the metallochaperone-like NmerA domain as the kinetically favored pathway in this co-evolved system.