The cyclophanes 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza-17,40-dioxa[10.1.10.1]paracyclophane (PO) and 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza[10.1.10.1]paracyclophane (PC) were coordinated with iron to form cationic binuclear Fe(III) Fe2PO and Fe2PC complexes, respectively. Their antioxidant capacity, superoxide dismutase and peroxidase mimetic activity, as well as their toxicity toward peripheral blood mononuclear cells (PBMCs) were evaluated. Both Fe2PO and Fe2PC are interesting biomimetics with antioxidant capacity similar to that of ascorbic acid that prevent mortality in cultured PBMCs, with the potential to have bioactive and protective functions in disease animal models.

Arginine kinase (AK) is a key enzyme for energetic balance in invertebrates. Although AK is a well-studied system that provides fast energy to invertebrates using the phosphagen phospho-arginine, the structural details on the AK-arginine binary complex interaction remain unclear. Herein, we determined two crystal structures of the Pacific whiteleg shrimp (Litopenaeus vannamei) arginine kinase, one in binary complex with arginine (LvAK-Arg) and a ternary transition state analog complex (TSAC). We found that the arginine guanidinium group makes ionic contacts with Glu225, Cys271 and a network of ordered water molecules. On the zwitterionic side of the amino acid, the backbone amide nitrogens of Gly64 and Val65 coordinate the arginine carboxylate. Glu314, one of proposed acid-base catalytic residues, did not interact with arginine in the binary complex. This residue is located in the flexible loop 310-320 that covers the active site and only stabilizes in the LvAK-TSAC. This is the first binary complex crystal structure of a guanidine kinase in complex with the guanidine substrate and could give insights into the nature of the early steps of phosphagen biosynthesis.

The compounds 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza-17,40-dioxa[10.1.10.1]paracyclophane and 2,9,25,32-tetraoxo-4,7,27,30-tetrakis(carboxymethyl)-1,4,7,10,24,27,30,33-octaaza[10.1.10.1]paracyclophane binuclear copper complexes (Cu2PO and Cu2PC, respectively) were studied by determining their antioxidant capacity using the TROLOX equivalent antioxidant capacity (TEAC) assay, and their cytotoxicity on cultured cells, as well as the superoxide dismutase (SOD)-like activity. Cu2PO had an antioxidant capacity (0.1 g eq TROLOX mol−1) within the order of magnitude of ascorbic acid, and both, Cu2PO and Cu2PC were nontoxic to cultured peripheral mononuclear blood cells. The SOD-like activity was evaluated using the nitroblue tetrazolium assay, and both compounds presented an excellent activity: for Cu2PO, the IC50 was 52 nM and for Cu2PC an IC50 of 0.5 μM was obtained comparable to CuZn SOD IC50 17 nM (Fernandes et al., J Inorg Biochem 2007;101:849–858). These results suggest that synthetic binuclear macrocycles are good candidates to be used as synthetic bioactive molecules with applications in biomedicine.

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH 2 THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen-bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k cat of the K48Q mutant was 430 fold lower than wild-type TS in activity, while the the K m for the (R)-stereoisomer of CH 2 THF was 300 µM, about 30 fold larger than K m from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideaza folate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutamt, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH 2 THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.

Biosynthesis of nucleoside triphosphates is critical for bioenergetics and nucleic acid replication, and this is achieved by nucleoside diphosphate kinase (NDK). As an emerging biological model and the global importance of shrimp culture, we have addressed the study of the Pacific whiteleg shrimp (Litopenaeus vannamei) NDK. We demonstrated its activity and affinity towards deoxynucleoside diphosphates. Also, the quaternary structure obtained by gel filtration chromatography showed that shrimp NDK is a trimer. Affinity was in the micro-molar range for dADP, dGDP, dTDP and except for dCDP, which presented no detectable interaction by isothermal titration calorimetry, as described previously for Plasmodium falciparum NDK. This information is particularly important, as this enzyme could be used to test nucleotide analogs that can block white spot syndrome virus (WSSV) viral replication and to study its bioenergetics role during hypoxia and fasting.

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With the objective of studying the conformational and macrocyclic effects of selected metal chelates on their peroxidase activities, Cu2+ and Fe3+ complexes were synthesized with a macrocyclic derivative of ethylenediaminetetraacetic acid and o-phenylenediamine (abbreviated as edtaodH2) and its new open-chain analogue (edtabzH2). The Fe3+ complex of edtaodH2 has a peroxidase-like activity, whereas the complex of edtabzH2 does not. The X-ray study of the former shows the formation of a dimeric molecule {[Fe(edtaod)]2O} in which each metal with an octahedral coordination is overposed over the macrocyclic cavity, as a result of rigid macrocyclic frame, to form an Fe–O–Fe bridge; the exposure of the central metal to the environment facilitates the capture of oxygen to drive the biomimetic activity. The peroxidase-inactive Fe3+ complex consists of a mononuclear complex ion [Fe(edtabz)(H2O)]+, the metal ion of which is suited in a distorted pentagonal bipyramid to be protected from environmental oxygen. The copper(II) complexes, which have mononuclear structures with high thermodynamic stability compared with the iron(III) complexes, show no peroxidase activity. The steric effects play a fundamental role in the biomimetic activity.

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