Cyclobutanone Inhibitors of Diaminopimelate Desuccinylase (DapE) as Potential New Antibiotics.

Based on our previous success in using cyclobutanone derivatives as enzyme inhibitors, we have designed and prepared a 37-member library of α-aminocyclobutanone amides and sulfonamides, screened for inhibition of the bacterial enzyme diaminopimelate desuccinylase (DapE), which is a promising antibiotic target, and identified several inhibitors with micromolar inhibitory potency. Molecular docking suggests binding of the deprotonated hydrate of the strained cyclobutanone, and thermal shift analysis with the most potent inhibitor (3y, IC50 = 23.1 µM) enabled determination of a Ki value of 10.2 +/- 0.26 µM and observed two separate Tm values for H. influenzae DapE (HiDapE).

Tetrazole-based inhibitors of the bacterial enzyme N-succinyl-l,l-2,6-diaminopimelic acid desuccinylase as potential antibiotics.

Based on a hit from a high-throughput screen, a series of phenyltetrazole amides was synthesized and assayed for inhibitory potency against DapE from Haemophilus influenzae (HiDapE). The inhibitory potency was modest but confirmed, with the most potent analog containing an aminothiazole moiety displaying an IC50 = 50.2 ± 5.0 µM. Docking reveals a potential binding mode wherein the amide carbonyl bridges both zinc atoms in the active site, and the tetrazole forms key hydrogen bonds with Arg330.

Synthesis and characterization of the N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) alternate substrate analog N,N-dimethyl-l,l-SDAP.

Growing antibiotic resistance by pathogenic bacteria has led to a global crisis. The bacterial enzyme N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) provides a very attractive target for the discovery of a new class of antibiotics, as it resides exclusively in many pathogenic bacterial strains and is a key enzyme in the lysine biosynthetic pathway. This pathway is responsible for the production of lysine as well as meso-diaminopimelate (m-DAP), both of which are required for peptidoglycan cell-wall synthesis, and lysine for peptide synthesis. The enzyme DapE catalyzes the hydrolysis of N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) to succinate and l,l-diaminopimelic acid (l,l-DAP), and due to its absence in humans, inhibition of DapE avoids mechanism-based side effects. We have executed the asymmetric synthesis of N,N-dimethyl-SDAP, an l,l-SDAP substrate analog and an analog of the synthetic substrate of our previously described DapE assay. Previous modeling studies advocated that N,N-dimethyl-SDAP might function as an inhibitor, however the compound behaves as a substrate, and we have demonstrated the use of N,N-dimethyl-SDAP as the substrate in a modified ninhydrin-based DapE assay. Thermal shift experiments of DapE in the presence of N,N-dimethyl-SDAP are consistent with a melt temperature (Tm) shifted by succinate, the product of enzymatic hydrolysis.

Atomic-Resolution 1.3 Å Crystal Structure, Inhibition by Sulfate, and Molecular Dynamics of the Bacterial Enzyme DapE.

We report the atomic-resolution (1.3 Å) X-ray crystal structure of an open conformation of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE, EC 3.5.1.18) from Neisseria meningitidis. This structure [Protein Data Bank (PDB) entry 5UEJ] contains two bound sulfate ions in the active site that mimic the binding of the terminal carboxylates of the N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) substrate. We demonstrated inhibition of DapE by sulfate (IC50 = 13.8 ± 2.8 mM). Comparison with other DapE structures in the PDB demonstrates the flexibility of the interdomain connections of this protein. This high-resolution structure was then utilized as the starting point for targeted molecular dynamics experiments revealing the conformational change from the open form to the closed form that occurs when DapE binds l,l-SDAP and cleaves the amide bond. These simulations demonstrated closure from the open to the closed conformation, the change in RMS throughout the closure, and the independence in the movement of the two DapE subunits. This conformational change occurred in two phases with the catalytic domains moving toward the dimerization domains first, followed by a rotation of catalytic domains relative to the dimerization domains. Although there were no targeting forces, the substrate moved closer to the active site and bound more tightly during the closure event.

"Inside-Out" PEGylation of Bovine ß-Cross-Linked Hemoglobin.

The development of a blood substitute is urgent due to blood shortages and potential communicable diseases. A novel method, inside-out PEGylation, has been used here to conjugate a multiarm maleimide-PEG (Mal-PEG) to ß-cross-linked (ßXL-Hb) hemoglobin (Hb) tetramers through the Cys ß93 residues. This method produces a polymer with a single PEG backbone that is surrounded by multiple proteins, rather than coating a single protein with multiple PEG chains. Electrophoresis under denaturing conditions showed a large molecular weight species. Gel filtration chromatography and analytical ultracentrifugation determined the most prevalent species had three ßXL-Hb to one Mal-PEG. Thermal denaturation studies showed that the cross-linked and PEGylated species were more stable than native Hb. Cross-linking under oxy-conditions produced a high oxygen affinity Hb species (P50  = 9.18 Torr), but the oxygen affinity was not significantly altered by PEGylation (P50  = 9.67 Torr). Inside-out PEGylation can be used to produce a hemoglobin-based oxygen carrier and potentially for other multiprotein complexes.

Unique mechanism of facile polymorphic conversion of acetaminophen in aqueous medium.

Rapid polymorphic conversion of acetaminophen (APAP) in solution, from metastable orthorhombic Form II to the stable monoclinic Form I, is well-known. The mechanism is believed to be solution-mediated phase transformation (SMPT), but with little experimental evidence. The present study was undertaken to understand this phenomenon from both thermodynamic and kinetic perspectives. Reliable apparent solubility of Form II was measured, for the first time, in 0.15 M aqueous NaCl solution at 37 °C. The solubility ratio of Form II over Form I, 1.27 ± 0.04, is quite low, which translates to a relatively low thermodynamic driving force for the conversion. Further solution crystallization experiments at supersaturation levels equal to or much greater than Form II solubility did not result in any crystallization in 10 days. Therefore, fast conversion is not possible through SMPT. To explore alternative mechanisms, molecular dynamics (MD) simulations were conducted to investigate the molecular level dissolution behavior and the solid state differences between the two polymorphs. The MD simulations reveal very different behavior. Form II exhibits a much higher rate of H-bond breakage, leading to the accumulation of a large number of disordered APAP molecules on the crystal surface. This thick disordered molecular layer provides a high local acetaminophen concentration which could be responsible for the fast crystallization of Form I. This was further supported by the observations made, using polarized light microscopy and powder X-ray diffractometry, when monitoring Form II crystals coming into contact with NaCl solution. We thus concluded that the hydrated surface layer is the "catalyst" for the facile phase conversion. This new mechanism, termed as SurFPT (surface-facilitated phase transformation), is much more effective in promoting polymorphic transformation than the well-known SMPT.

Unsuspected pathway of the allosteric transition in hemoglobin.

Large conformational transitions play an essential role in the function of many proteins, but experiments do not provide the atomic details of the path followed in going from one end structure to the other. For the hemoglobin tetramer, the transition path between the unliganded (T) and tetraoxygenated (R) structures is not known, which limits our understanding of the cooperative mechanism in this classic allosteric system, where both tertiary and quaternary changes are involved. The conjugate peak refinement algorithm is used to compute an unbiased minimum energy path at atomic detail between the two end states. Although the results confirm some of the proposals of Perutz [Perutz MF (1970) Stereochemistry of cooperative effects in haemoglobin. Nature 228:726-734], the subunit motions do not follow the textbook description of a simple rotation of one αß-dimer relative to the other. Instead, the path consists of two sequential quaternary rotations, each involving different subdomains and axes. The quaternary transitions are preceded and followed by phases of tertiary structural changes. The results explain the recent photodissociation measurements, which suggest that the quaternary transition has a fast (2 µs) as well as a slow (20 µs) component and provide a testable model for single molecule FRET experiments.

N α -acetyl-L-ornithine deacetylase from Escherichia coli and a ninhydrin-based assay to enable inhibitor identification.

Bacteria are becoming increasingly resistant to antibiotics, therefore there is an urgent need for new classes of antibiotics to fight antibiotic resistance. Mammals do not express N ɑ -acetyl-L-ornithine deacetylase (ArgE), an enzyme that is critical for bacterial survival and growth, thus ArgE represents a promising new antibiotic drug target, as inhibitors would not suffer from mechanism-based toxicity. A new ninhydrin-based assay was designed and validated that included the synthesis of the substrate analog N 5, N 5-di-methyl N α-acetyl-L-ornithine (kcat/Km = 7.32 ± 0.94 × 104 M-1s-1). This new assay enabled the screening of potential inhibitors that absorb in the UV region, and thus is superior to the established 214 nm assay. Using this new ninhydrin-based assay, captopril was confirmed as an ArgE inhibitor (IC50 = 58.7 µM; Ki = 37.1 ± 0.85 µM), and a number of phenylboronic acid derivatives were identified as inhibitors, including 4-(diethylamino)phenylboronic acid (IC50 = 50.1 µM). Selected inhibitors were also tested in a thermal shift assay with ArgE using SYPRO Orange dye against Escherichia coli ArgE to observe the stability of the enzyme in the presence of inhibitors (captopril Ki = 35.9 ± 5.1 µM). The active site structure of di-Zn EcArgE was confirmed using X-ray absorption spectroscopy, and we reported two X-ray crystal structures of E. coli ArgE. In summary, we describe the development of a new ninhydrin-based assay for ArgE, the identification of captopril and phenylboronic acids as ArgE inhibitors, thermal shift studies with ArgE + captopril, and the first two published crystal structures of ArgE (mono-Zn and di-Zn).

Biochemical and Structural Analysis of the Bacterial Enzyme Succinyl-Diaminopimelate Desuccinylase (DapE) from Acinetobacter baumannii.

There is an urgent need for new antibiotics given the rise of antibiotic resistance, and succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) has emerged as a promising bacterial enzyme target. DapE from Haemophilus influenzae (HiDapE) has been studied and inhibitors identified, but it is essential to explore DapE from different species to assess selective versus broad-spectrum therapeutics. We have determined the structure of DapE from the ESKAPE pathogen Acinetobacter baumannii (AbDapE) and studied inhibition by known inhibitors of HiDapE. AbDapE is inhibited by captopril and sulfate comparable to HiDapE, but AbDapE was not significantly inhibited by a known indoline sulfonamide HiDapE inhibitor. Captopril and sulfate both stabilize HiDapE by increasing the thermal melting temperature (Tm) in thermal shift assays. By contrast, sulfate decreases the stability of the AbDapE enzyme, whereas captopril increases the stability. Further, we report two crystal structures of selenomethionine-substituted AbDapE in the closed conformation, one with AbDapE in complex with succinate derived from enzymatic hydrolysis of N6-methyl-l,l-SDAP substrate and acetate (PDB code 7T1Q, 2.25 Å resolution), and a crystal structure of AbDapE with bound succinate along with l-(S)-lactate, a product of degradation of citric acid from the crystallization buffer during X-ray irradiation (PDB code 8F8O, 2.10 Å resolution).

Molecular dynamics of drug crystal dissolution: simulation of acetaminophen form I in water.

In order to gain molecular level understanding of drug dissolution into aqueous media, we report the first molecular dynamics (MD) simulation of a drug crystal dissolving. The simulation was performed for acetaminophen crystal Form I dissolving in 0.15 M aqueous NaCl solution at 37 °C. The 10 ns simulation revealed interesting details of the dissolution process. Dissolution of the molecules from the crystal surface is far from a random process. On the contrary, the order in which molecules enter the solution depends on their initial positions in or on the crystal. Molecules located on the corners and edges dissolved first followed by those located on {100}, {010}, and {001) surfaces with slight variation. This corner and edge effect that has been observed in our real dissolution experiment conducted under polarized light microscope was successfully predicted at molecular scale by the MD simulation. Further analyses identified the underlying mechanism: the differences in the molecular interaction energetics between the drug and water molecules. The molecules located on corners and edges of the parallelepiped crystal are not as tightly bound to their surrounding neighbors as those located in other positions, but they are more strongly interacting with the surrounding water molecules. The extent of molecular release is strongly correlated with the interplay between interaction forces with solvent molecules and with other drug molecules in the crystal lattice. These findings, especially the significant "corner and edge effect", will help us gain additional fundamental understanding in the relationship between dissolution rate and particle size and morphology and, thus, are very relevant in the context of particle size reduction in delivering poorly water-soluble compounds. This study has also demonstrated that MD simulation is a powerful tool in studying dissolution phenomena.

Ligand migration in the truncated hemoglobin of Mycobacterium tuberculosis.

The truncated hemoglobin of Mycobacterium tuberculosis (Mt-trHbO) is a small heme protein belonging to the hemoglobin superfamily. Truncated hemoglobins (trHbs) are believed to have functional roles such as terminal oxidases and oxygen sensors involved in the response to oxidative and nitrosative stress, nitric oxide (NO) detoxification, O2/NO chemistry, O2 delivery under hypoxic conditions, and long-term ligand storage. Based on sequence similarities, they are classified into three groups. Experimental studies revealed that all trHbs display a 2-on-2 α-helical sandwich fold rather than the 3-on-3 α-helical sandwich fold of the classical hemoglobin fold. Using locally enhanced sampling (LESMD) molecular dynamics, the ligand-binding escape pathways from the distal heme binding cavity of Mt-trHbO were determined to better understand how this protein functions. The importance of specific residues, such as the group II and III invariant W(G8) residue, can be seen in terms of ligand diffusion pathways and ligand dynamics. LESMD simulations show that the wild-type Mt-trHbO has three diffusion pathways while the W(G8)F Mt-trHbO mutant has only two. The W(G8) residue plays a critical role in ligand binding and stabilization and helps regulate the rate of ligand escape from the distal heme pocket. Thus, this invariant residue is important in creating ligand diffusion pathways and possibly in the enzymatic functions of this protein.

Menadione degrades the optical quality and mitochondrial integrity of bovine crystalline lenses.

PURPOSE: The crystalline lens is a unique cellular organ that performs metabolic processes while maintaining transparency for optical functionality. Mitochondria play a role in providing cells with aerobic respiration necessary for these metabolic processes. Using menadione, a mitochondria-specific inhibitor of the quinone family, and bovine lenses in vitro, this study was undertaken to determine whether a relationship exists between mitochondrial function and optical function. METHODS: Bovine lenses were treated with 50 µM, 200 µM, 600 µM, and 1,000 µM menadione and lens optical function, assessed as optical quality, was observed over 9 days. Confocal micrographs of mitochondria in superficial secondary fiber cells were also analyzed in 50 µM, 200 µM, and 600 µM menadione-treated lenses over 48 h. RESULTS: A decrease in lens optical quality was observed in a dose-dependent manner within 24 h for the 200 µM- (p=0.0422), 600 µM- (p<0.0001), and 1,000 µM- (p<0.0001) treated lenses. No change in optical quality was observed for the 50 µM-treated lenses. Analysis of confocal micrographs indicated a trend of shorter mitochondria for 200 µM- and 600 µM-treated lenses with time and analysis of the distributions of mitochondrial lengths indicated a relative increase in the number of shorter mitochondria with higher doses of, and longer exposures to, menadione. CONCLUSIONS: The data show that menadione has a detrimental effect on mitochondrial integrity and this change is associated with degradation of optical quality, suggesting a possible link between mitochondrial function and optical function.

Effect of cross-linker length on the stability of hemoglobin.

A series of cross-linking reagents with 4 to 7 carbons have been synthesized and used to modify human hemoglobin. The product yields and biochemical properties of these cross-linked hemoglobins are compared to those made with both longer and shorter cross-linkers. Several trends become apparent. The yields decrease as the cross-linker becomes longer, which correlates well with molecular dynamics studies of reagent binding pathways presented here. The autooxidation rates increase while thermal stability decreases with longer reagents. Cross-linking under deoxy conditions also increases autooxidation rates, but the effect is less than that of increased cross-linker length. The results suggest that shorter reagents may provide better-stabilized tetramers for the construction of more complex hemoglobin-based oxygen carriers.

Mapping of a Regulatory Site of the Escherichia coli ADP-Glucose Pyrophosphorylase.

The enzyme ADP-glucose pyrophosphorylase (ADP-Glc PPase) controls the biosynthesis of glycogen in bacteria and starch in plants. It is regulated by various activators in different organisms according to their metabolic characteristics. In Escherichia coli, the major allosteric activator is fructose 1,6-bisphosphate (FBP). Other potent activator analogs include 1,6-hexanediol bisphosphate (HBP) and pyridoxal 5'-phosphate (PLP). Recently, a crystal structure with FBP bound was reported (PDB ID: 5L6S). However, it is possible that the FBP site found is not directly responsible for the activation of the enzyme. We hypothesized FBP activates by binding one of its phosphate groups to another site ("P1") in which a sulfate molecule was observed. In the E. coli enzyme, Arg40, Arg52, and Arg386 are part of this "P1" pocket and tightly complex this sulfate, which is also present in the crystal structures of ADP-Glc PPases from Agrobacterium tumefaciens and Solanum tuberosum. To test this hypothesis, we modeled alternative binding conformations of FBP, HBP, and PLP into "P1." In addition, we performed a scanning mutagenesis of Arg residues near potential phosphate binding sites ("P1," "P2," "P3"). We found that Arg40 and Arg52 are essential for FBP and PLP binding and activation. In addition, mutation of Arg386 to Ala decreased the apparent affinity for the activators more than 35-fold. We propose that the activator binds at this "P1" pocket, as well as "P2." Arg40 and Arg52 are highly conserved residues and they may be a common feature to complex the phosphate moiety of different sugar phosphate activators in the ADP-Glc PPase family.

Use of the conjugate peak refinement algorithm for identification of ligand-binding pathways in globins.

Determination of the three-dimensional structures of the globins led to the problem of determining how the ligands bound to the heme. In many of these structures there was no clear path from the solvent to the ligand-binding site. Even in those structures that appeared to have one or more tunnels from the exterior to the heme, it was not clear that these were the only paths for ligand access. Conjugate peak refinement (CPR) is a computational technique that can provide a minimum energy path between two conformations of a protein. By defining one conformation as the unbound structure with an external ligand and the other as the ligand-bound structure, CPR can be used to determine a pathway for ligand binding.

Identification of ligand-binding pathways in truncated hemoglobins using locally enhanced sampling molecular dynamics.

This chapter reviews the use of a locally enhanced sampling molecular dynamics (LESMD) for the study of ligand binding in truncated hemoglobins. The method, however, can be applied to any protein-ligand system. Truncated hemoglobins appear to have a tunnel(s) potentially used by the ligand to bind. These structural features give some indication of how the ligand moves through the protein to bind but do not give the complete picture. The LESMD method has been used to investigate the pathways of ligand binding to group I truncated hemoglobins from the eubacteria Mycobacterium tuberculosis, the ciliated protozoa Paramecium caudatum, and the unicellular alga Chlamydomonas eugametos.

Finding gas migration pathways in proteins using implicit ligand sampling.

Implicit ligand sampling is a practical, efficient, and accurate method for finding the gas migration pathways for small hydrophobic gas molecules, such as oxygen, inside proteins. The method infers the gas migration pathways by calculating the potential of mean force for the gas molecule everywhere inside the protein by means of a molecular dynamics simulation of the protein in the absence of the gas molecule. Pathways can be constructed by connecting the areas of the protein that are favorable to the presence of gas. This method has the advantage of providing a comprehensive overview of all possible gas migration pathways and barriers in a given protein from a single simulation run. Implicit ligand sampling has been applied to a large number of hemoproteins. The example of the truncated hemoglobin from Paramecium caudatum is given to illustrate the method.

Spectroelectrochemical study of hemoglobin A, alpha- and beta-fumarate crosslinked hemoglobins; implications to autoxidation reaction.

The thermodynamics and kinetics of the reaction DeoxyHb-Fe(2+)<-->MetHb-Fe(3+) for human hemoglobin A (HbA), alpha- and beta-fumarate crosslinked hemoglobins were investigated by spectroelectrochemistry. Information from this study is used to determine what structural features and experimental conditions stabilize ferrous vs. ferric form of hemoglobin, and what implications this stabilization may have on the autoxidation reaction. Alpha- and beta-fumarate crosslinked hemoglobins, alphaXL-HbA and betaXL-HbA, were obtained by crosslinking deoxyhemoglobin and oxyhemoglobin, respectively, with bis(3,5-dibromosalicyl) fumarate (DBSF). Formal redox potentials, E(0), and reduction/oxidation rates were measured in the presence of mediator, hexammineruthenium(III) chloride. It was found that E(0) shifted positive for the alpha-, and negative for the beta-fumarate crosslinked hemoglobin compared to HbA for all experimental conditions investigated. This shift was consistent with stabilization of the tense (positive shift) or relaxed conformation (negative shift) conferred by crosslinking. Formal redox potentials shifted positive with addition of nitrate and chloride ions for alphaXL-HbA, indicating additional stabilization of the T quaternary. The slopes of the Nernst plots showed evidence of cooperativity as expressed by n(max). The data points (E(0), n(max)) were fitted by the MWC model which states that the electron transfer and the addition/removal of water are concerted. The set of K(R) and c values, where the parameter c is the ratio K(R)/K(T) and K(R) and K(T) are the ligand (water molecule and an electron-hole) dissociation constants for the R and T states, for the beta-crosslinked hemoglobin compared to that of HbA and alpha-crosslinked hemoglobin indicated that crosslinking of oxyhemoglobin affected differently the inner-coordination sphere at the heme site. By modulating the electrolyte concentration the reduction rates were measured as a function of DeltaE(0), the difference in E(0) between hemoglobin molecules and mediator. Linearization of the Marcus cross-relationship (based on the concerted water and electron transfer) was good for HbA, and poor for alphaXL-HbA and betaXL-HbA, consistent with results obtained by the MWC analysis. This may imply that the reduction of HbA is controlled by the driving force, DeltaE(0), whereas the reduction of alphaXL-HbA and betaXL-HbA occurs by a non-concerted mechanism controlled by structural features brought about by crosslinking. The autoxidation reaction, conversion of oxygen-bound ferrous hemoglobin to ferric hemoglobin, was found independent of E(0). Alpha-fumarate crosslinked hemoglobin showed the highest autoxidation rate despite its positive shift in formal redox potential as compared to HbA, followed by beta-fumarate crosslinked hemoglobin, and by native hemoglobin. These data suggest that the chemical mechanism of oxygen dissociation and accessibility of water and oxygen radicals to heme site control autoxidation.

Folding of Gα Subunits: Implications for Disease States.

G-proteins play a central role in signal transduction by fluctuating between "on" and "off" phases that are determined by a conformational change. cAMP is a secondary messenger whose formation is inhibited or stimulated by activated Giα1 or Gsα subunit. We used tryptophan fluorescence, UV/vis spectrophotometry, and circular dichroism to probe distinct structural features within active and inactive conformations from wild-type and tryptophan mutants of Giα1 and Gsα. For all proteins studied, we found that the active conformations were more stable than the inactive conformations, and upon refolding from higher temperatures, activated wild-type subunits recovered significantly more native structure. We also observed that the wild-type subunits partially regained the ability to bind nucleotide. The increased compactness observed upon activation was consistent with the calculated decrease in solvent accessible surface area for wild-type Giα1. We found that as the temperature increased, Gα subunits, which are known to be rich in α-helices, converted to proteins with increased content of ß-sheets and random coil. For active conformations from wild-type and tryptophan mutants of Giα1, melting temperatures indicated that denaturation starts around hydrophobic tryptophan microenvironments and then radiates toward tyrosine residues at the surface, followed by alteration of the secondary structure. For Gsα, however, disruption of secondary structure preceded unfolding around tyrosine residues. In the active conformations, a π-cation interaction between essential arginine and tryptophan residues, which was characterized by a fluorescence-measured red shift and modeled by molecular dynamics, was also shown to be a contributor to the stability of Gα subunits. The folding properties of Gα subunits reported here are discussed in the context of diseases associated to G-proteins.

Characterization of a protozoan Phosducin-like protein-3 (PhLP-3) reveals conserved redox activity.

We recently identified three novel thioredoxin-like genes in the genome of the protozoan parasite Plasmodium that belong to the Phosducin-like family of proteins (PhLP). PhLPs are small cytosolic proteins hypothesized to function in G-protein signaling and protein folding. Although PhLPs are highly conserved in eukaryotes from yeast to mammals, only a few representatives have been experimentally characterized to date. In addition, while PhLPs contain a thioredoxin domain, they lack a CXXC motif, a strong indicator for redox activity, and it is unclear whether members of the PhLP family are enzymatically active. Here, we describe PbPhLP-3 as the first phosducin-like protein of a protozoan organism, Plasmodium berghei. Initial transcription analysis revealed continuous low-level expression of pbphlp-3 throughout the complex Plasmodium life cycle. Attempts to knockout pbphlp-3 in P. berghei did not yield live parasites, suggesting an essential role for the gene in Plasmodium. We cloned, expressed and purified PbPhLP-3 and determined that the recombinant protein is redox active in vitro in a thioredoxin-coupled redox assay. It also has the capacity to reduce the organic compound tert-Butyl hydroperoxide (TBHP) in vitro, albeit at low efficiency. Sequence analysis, structural modeling, and site-directed mutagenesis revealed a conserved cysteine in the thioredoxin domain to be the redox active residue. Lastly, we provide evidence that recombinant human PhLP-3 exhibits redox activity similar to that of PbPhLP-3 and suggest that redox activity may be conserved in PhLP-3 homologs of other species. Our data provide new insight into the function of PhLP-3, which is hypothesized to act as co-chaperones in the folding and regulation of cytoskeletal proteins. We discuss the potential implications of PhLP-3 as a thioredoxin-target protein and possible links between the cellular redox network and the eukaryotic protein folding machinery.