The B800-820, or LH3, complex is a spectroscopic variant of the B800-850 LH2 peripheral light-harvesting complex. LH3 is synthesized by some species and strains of purple bacteria when growing under what are generally classed as "stressed" conditions, such as low intensity illumination and/or low temperature (<30 degrees C). The apoproteins in these complexes modify the absorption properties of the chromophores to ensure that the photosynthetic process is highly efficient. The crystal structure of the B800-820 light-harvesting complex, an integral membrane pigment-protein complex, from the purple bacteria Rhodopseudomonas (Rps.) acidophila strain 7050 has been determined to a resolution of 3.0 A by molecular replacement. The overall structure of the LH3 complex is analogous to that of the LH2 complex from Rps. acidophila strain 10050. LH3 has a nonameric quaternary structure where two concentric cylinders of alpha-helices enclose the pigment molecules bacteriochlorophyll a and carotenoid. The observed spectroscopic differences between LH2 and LH3 can be attributed to differences in the primary structure of the apoproteins. There are changes in hydrogen bonding patterns between the coupled Bchla molecules and the protein that have an effect on the conformation of the C3-acetyl groups of the B820 molecules. The structure of LH3 shows the important role that the protein plays in modulating the characteristics of the light-harvesting system and indicates the mechanisms by which the absorption properties of the complex are altered to produce a more efficient light-harvesting component.

Pteridine reductase (PTR1) is essential for salvage of pterins by parasitic trypanosomatids and is a target for the development of improved therapies. To identify inhibitors of Leishmania major and Trypanosoma cruzi PTR1, we combined a rapid-screening strategy using a folate-based library with structure-based design. Assays were carried out against folate-dependent enzymes including PTR1, dihydrofolate reductase (DHFR), and thymidylate synthase. Affinity profiling determined selectivity and specificity of a series of quinoxaline and 2,4-diaminopteridine derivatives, and nine compounds showed greater activity against parasite enzymes compared with human enzymes. Compound 6a displayed a Ki of 100 nM toward LmPTR1, and the crystal structure of the LmPTR1:NADPH:6a ternary complex revealed a substrate-like binding mode distinct from that previously observed for similar compounds. A second round of design, synthesis, and assay produced a compound (6b) with a significantly improved Ki (37 nM) against LmPTR1, and the structure of this complex was also determined. Biological evaluation of selected inhibitors was performed against the extracellular forms of T. cruzi and L. major, both wild-type and overexpressing PTR1 lines, as a model for PTR1-driven antifolate drug resistance and the intracellular form of T. cruzi. An additive profile was observed when PTR1 inhibitors were used in combination with known DHFR inhibitors, and a reduction in toxicity of treatment was observed with respect to administration of a DHFR inhibitor alone. The successful combination of antifolates targeting two enzymes indicates high potential for such an approach in the development of previously undescribed antiparasitic drugs.antitrypanosomatid agents ͉ antifolates ͉ drug discovery P rotozoan parasites of the order Kinetoplastida are the causal agents of serious human diseases, including African sleeping sickness, Chagas' disease, and leishmaniasis. There is an urgent need for new, more effective drugs targeting these neglected diseases, because those in current use are toxic, expensive, and often difficult to administer. The problem is compounded by an increase in drug resistance and lack of progress in drug development. Only a single new effective treatment has been developed in the last 25 years, Miltefosine (hexadecylphosphocholine), recently approved in India (1).Enzymes involved in the provision and use of reduced folate cofactors such as dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are valued drug targets for the treatment of bacterial infections (2), cancer (3), and certain parasitic diseases, notably malaria (4). DHFR catalyzes the two-step reduction of folate to tetrahydrofolate, which is then transformed to N 5 ,N 10 -methylene tetrahydrofolate and is used by TS as a methyl donor and reducing agent in the conversion of 2Ј-deoxyuridine-5Ј-monophosphate to 2Ј-deoxythymidine-5Ј-monophosphate. Inhibition of DHFR or TS reduces the cellular pool of 2Ј-deoxythymidine-5Ј-monophosphate, impairing DNA replication and resulting ...

SummaryThe protozoan Trypanosoma brucei has a functional pteridine reductase (TbPTR1), an NADPH-dependent short-chain reductase that participates in the salvage of pterins, which are essential for parasite growth. PTR1 displays broad-spectrum activity with pterins and folates, provides a metabolic bypass for inhibition of the trypanosomatid dihydrofolate reductase and therefore compromises the use of antifolates for treatment of trypanosomiasis. Catalytic properties of recombinant TbPTR1 and inhibition by the archetypal antifolate methotrexate have been characterized and the crystal structure of the ternary complex with cofactor NADP + and the inhibitor determined at 2.2 Å resolution. This enzyme shares 50% amino acid sequence identity with Leishmania major PTR1 (LmPTR1) and comparisons show that the architecture of the cofactor binding site, and the catalytic centre are highly conserved, as are most interactions with the inhibitor. However, specific amino acid differences, in particular the placement of Trp221 at the side of the active site, and adjustment of the b6-a6 loop and a6 helix at one side of the substrate-binding cleft significantly reduce the size of the substrate binding site of TbPTR1 and alter the chemical properties compared with LmPTR1. A reactive Cys168, within the active site cleft, in conjunction with the C-terminus carboxyl group and His267 of a partner subunit forms a triad similar to the catalytic component of cysteine proteases. TbPTR1 therefore offers novel structural features to exploit in the search for inhibitors of therapeutic value against African trypanosomiasis.

Metacaspases are distantly related caspase-family cysteine peptidases implicated in programmed cell death in plants and lower eukaryotes. They differ significantly from caspases because they are calcium-activated, arginine-specific peptidases that do not require processing or dimerization for activity. To elucidate the basis of these differences and to determine the impact they might have on the control of cell death pathways in lower eukaryotes, the previously undescribed crystal structure of a metacaspase, an inactive mutant of metacaspase 2 (MCA2) from Trypanosoma brucei, has been determined to a resolution of 1.4 Å. The structure comprises a core caspase fold, but with an unusual eight-stranded β-sheet that stabilizes the protein as a monomer. Essential aspartic acid residues, in the predicted S1 binding pocket, delineate the arginine-specific substrate specificity. In addition, MCA2 possesses an unusual N terminus, which encircles the protein and traverses the catalytic dyad, with Y31 acting as a gatekeeper residue. The calcium-binding site is defined by samarium coordinated by four aspartic acid residues, whereas calcium binding itself induces an allosteric conformational change that could stabilize the active site in a fashion analogous to subunit processing in caspases. Collectively, these data give insights into the mechanistic basis of substrate specificity and mode of activation of MCA2 and provide a detailed framework for understanding the role of metacaspases in cell death pathways of lower eukaryotes.apoptosis | clan CD | parasite | X-ray crystallography P rogrammed cell death (PCD) is essential for animal development and the maintenance of adult tissues. PCD itself is a regulated process, and in animals, apoptosis is controlled through the action of caspases, aspartic acid-specific cysteine peptidases (1). PCD is also essential for plant development (2), and multiple markers for apoptosis have been described in yeast and a broad range of protozoan parasites (3), yet these organisms do not encode caspases in their genomes; thus, alternative pathways must have evolved for caspase-independent cell death to be regulated. In recent years, attention has focused on the metacaspases, which are a highly conserved group of caspase-like cysteine peptidases found in plants, fungi, and protozoa but not in the metazoa (4). In plants, metacaspases are essential for embryogenesis (5, 6) and have been shown to act in antagonistic relationships, functioning as both positive and negative regulators of PCD (7). In yeast and some protozoa, metacaspases have been implicated as mediators of cell death in response to oxidative stress and environmental change (3,(8)(9)(10).Various attempts have been made to draw parallels between caspases and metacaspases in respect to structure, activation, and function (11). However, two striking differences between metacaspases and caspases are their substrate specificities and requirements for activation. Metacaspases have arginine/lysine specificity, do not necessarily require proces...