Pteridine reductase (PTR1) is a short-chain reductase (SDR) responsible for the salvage of pterins in parasitic trypanosomatids. PTR1 catalyzes the NADPH-dependent two-step reduction of oxidized pterins to the active tetrahydro-forms and reduces susceptibility to antifolates by alleviating dihydrofolate reductase (DHFR) inhibition. Crystal structures of PTR1 complexed with cofactor and 7,8-dihydrobiopterin (DHB) or methotrexate (MTX) delineate the enzyme mechanism, broad spectrum of activity and inhibition by substrate or an antifolate. PTR1 applies two distinct reductive mechanisms to substrates bound in one orientation. The first reduction uses the generic SDR mechanism, whereas the second shares similarities with the mechanism proposed for DHFR. Both DHB and MTX form extensive hydrogen bonding networks with NADP(H) but differ in the orientation of the pteridine.

The molybdate-dependent transcriptional regulator (ModE) from Escherichia coli functions as a sensor of molybdate concentration and a regulator for transcription of operons involved in the uptake and utilization of the essential element, molybdenum. We have determined the structure of ModE using multi-wavelength anomalous dispersion. Selenomethionyl and native ModE models are refined to 1.75 and 2.1 Å, respectively and describe the architecture and structural detail of a complete transcriptional regulator. ModE is a homodimer and each subunit comprises N-and C-terminal domains. The N-terminal domain carries a winged helix-turn-helix motif for binding to DNA and is primarily responsible for ModE dimerization. The C-terminal domain contains the molybdate-binding site and residues implicated in binding the oxyanion are identified. This domain is divided into sub-domains a and b which have similar folds, although the organization of secondary structure elements varies. The sub-domain fold is related to the oligomer binding-fold and similar to that of the subunits of several toxins which are involved in extensive protein-protein interactions. This suggests a role for the C-terminal domain in the formation of the ModE-protein-DNA complexes necessary to regulate transcription. Modelling of ModE interacting with DNA suggests that a large distortion of DNA is not necessary for complex formation.

Tryparedoxin-I is a recently discovered thiol-disulfide oxidoreductase involved in the regulation of oxidative stress in parasitic trypanosomatids. The crystal structure of recombinant Crithidia fasciculata tryparedoxin-I in the oxidized state has been determined using multi-wavelength anomalous dispersion methods applied to a selenomethionyl derivative. The model comprises residues 3 to 145 with 236 water molecules and has been refined using all data between a 19-and 1.4-Å resolution to an R-factor and R-free of 19.1 and 22.3%, respectively. Despite sharing only about 20% sequence identity, tryparedoxin-I presents a five-stranded twisted ␤-sheet and two elements of helical structure in the same type of fold as displayed by thioredoxin, the archetypal thiol-disulfide oxidoreductase. However, the relationship of secondary structure with the linear amino acid sequences is different for each protein, producing a distinctive topology. The ␤-sheet core is extended in the trypanosomatid protein with an N-terminal ␤-hairpin. There are also differences in the content and orientation of helical elements of secondary structure positioned at the surface of the proteins, which leads to different shapes and charge distributions between human thioredoxin and tryparedoxin-I. A righthanded redox-active disulfide is formed between Cys-40 and Cys-43 at the N-terminal region of a distorted ␣-helix (␣1). Cys-40 is solvent-accessible, and Cys-43 is positioned in a hydrophilic cavity. Three C-H⅐⅐⅐O hydrogen bonds donated from two proline residues serve to stabilize the disulfide-carrying helix and support the correct alignment of active site residues. The accurate model for tryparedoxin-I allows for comparisons with the family of thiol-disulfide oxidoreductases and provides a template for the discovery or design of selective inhibitors of hydroperoxide metabolism in trypanosomes. Such inhibitors are sought as potential therapies against a range of human pathogens.Parasitic trypanosomatids, belonging to the order Kinetoplastida, cause debilitating and life-threatening human diseases such as African sleeping sickness, Chagas' disease, and the leishmaniases (1). The current therapies against these infections are inadequate due to poor drug efficacy and toxicity combined with increasing drug resistance (2). There is therefore an urgent need to understand how drugs already in use function so that they might be improved and to identify new targets for chemotherapeutic attack. The ideal target is an enzyme of a metabolic pathway that is essential for the survival of the parasite and either absent in the human host or one that presents differing substrate specificities (3). Because trypanosomatids are susceptible to oxidative stress, this aspect of their metabolism represents an attractive target for the development of new trypanocidal agents (4 -6).As with other organisms living in an aerobic environment, trypanosomes are exposed to reactive oxygen intermediates such as superoxide anion and hydrogen peroxide. These potentially destructive c...

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The molybdate-dependent transcriptional regulator ModE of Escherichia coli functions as a sensor of intracellular molybdate concentration and a regulator for the transcription of several operons that control the uptake and utilization of molybdenum. We present two high-resolution crystal structures of the C-terminal oxyanion-binding domain in complex with molybdate and tungstate. The ligands bind between subunits at the dimerization interface, and analysis reveals that oxyanion selectivity is determined primarily by size. The relevance of the structures is indicated by fluorescence measurements, which show that the oxyanion binding properties of the C-terminal domain of ModE are similar to those of the full-length protein. Comparisons with the apoprotein structure have identified structural rearrangements that occur on binding oxyanion. This molybdate-dependent conformational switch promotes a change in shape and alterations to the surface of the protein and may provide the signal for recruitment of other proteins to construct the machinery for transcription. Sequence and structure-based comparisons lead to a classification of molybdate-binding proteins.Molybdenum is an essential trace element required for the catalytic activity of several enzymes in animals, plants, and bacteria. In some cases the transition metal is complexed with a unique pterin forming the molybdopterin cofactor, and in others it forms part of an iron-molybdenum cluster cofactor (1, 2). Escherichia coli acquires molybdenum in the form of MoO 4 2Ϫ by using a high-affinity ABC-type molybdate transporter system encoded by the modABCD operon (3). ModA, the structure of which has been determined (4, 5), is similar to the sulfatebinding protein of Salmonella typhimurium (SBP) 1 (6) and a member of the periplasmic-binding protein family. ModA binds and transfers molybdate to ModB at the outer surface of the cytoplasmic membrane. ModB is an integral membrane protein and ModC a membrane associated protein; together they transport the oxyanion across the membrane using an ATP-dependent mechanism. The role of ModD is at present unknown. Exposure of E. coli to high levels of molybdate leads to repression of modABCD by a mechanism that involves the molybdatedependent transcriptional regulator known as ModE (7-9). The modE gene, situated immediately upstream from the mod-ABCD operon, codes for a transcriptional regulator able to control the uptake and utilization of this transition metal. ModE binds molybdate, and the complex can function as a repressor by binding at a site that overlaps the transcription start of the modABCD operon (10 -12). In addition, the ModEmolybdate complex acts as a positive regulator of several genes including those coding for the molybdoenzymes dimethyl sulfoxide reductase (13) and nitrate reductase A (14), as well as for hyc, the hydrogenase 3 structural operon. It also mediates expression of the moaABCDE operon, which encodes the first enzymes required for the biosynthesis of molybdopterin (15).E. coli ModE functions as a ho...

The dodecameric type II dehydroquinases (DHQases) have an unusual quaternary structure in which four trimeric units are arranged with cubic 23 symmetry. The unfolding and refolding behaviour of the enzymes from Streptomyces coelicolor and Mycobacterium tuberculosis have been studied. Gel-permeation studies show that, at low concentrations (0.5 M) of guanidinium chloride (GdmCl), both enzymes dissociate into trimeric units, with little or no change in the secondary or tertiary structure and with a 15% loss (S. coelicolor) or a 55% increase (M. tuberculosis) in activity. At higher concentrations of GdmCl, both enzymes undergo sharp unfolding transitions over narrow ranges of the denaturant concentration, consistent with co-operative unfolding of the subunits. When the concentration of GdmCl is lowered by dilution from 6 M to 0.55 M, the enzyme from S. coelicolor refolds in an efficient manner to form trimeric units, with more than 75% regain of activity. Using a similar approach the M. tuberculosis enzyme regains less than 35% activity. From the time courses of the changes in CD, fluorescence and activity of the S. coelicolor enzyme, an outline model for the refolding of the enzyme has been proposed. The model involves a rapid refolding event in which approximately half the secondary structure is regained. A slower folding process follows within the monomer, resulting in acquisition of the full secondary structure. The major changes in fluorescence occur in a second-order process which involves the association of two folded monomers. Regain of activity is dependent on a further associative event, showing that the minimum active unit must be at least trimeric. Reassembly of the dodecameric S. coelicolor enzyme and essentially complete regain of activity can be accomplished if the denatured enzyme is dialysed extensively to remove GdmCl. These results are discussed in terms of the recently solved X-ray structures of type II DHQases from these sources.

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The structures of enzymes catalyzing the reactions in central metabolic pathways are generally well conserved as are their catalytic mechanisms. The two types of 3-dehydroquinate dehydratase (DHQase) are therefore most unusual since they are unrelated at the sequence level and they utilize completely different mechanisms to catalyze the same overall reaction. The type I enzymes catalyze a cis-dehydration of 3-dehydroquinate via a covalent imine intermediate, while the type II enzymes catalyze a trans-dehydration via an enolate intermediate. Here we report the three-dimensional structures of a representative member of each type of biosynthetic DHQase. Both enzymes function as part of the shikimate pathway, which is essential in microorganisms and plants for the biosynthesis of aromatic compounds including folate, ubiquinone and the aromatic amino acids. An explanation for the presence of two different enzymes catalyzing the same reaction is presented. The absence of the shikimate pathway in animals makes it an attractive target for antimicrobial agents. The availability of these two structures opens the way for the design of highly specific enzyme inhibitors with potential importance as selective therapeutic agents.