Key substrate recognition residues in the active site of cystathionine beta-synthase from Toxoplasma gondii.

Cystathionine ß-synthase (CBS) catalyzes the condensation of l-serine and l-homocysteine to give l-cystathionine in the transsulfuration pathway. Recently, a few O-acetylserine (l-OAS)-dependent CBSs (OCBSs) have been found in bacteria that can exclusively function with l-OAS. CBS from Toxoplasma gondii (TgCBS) can efficiently use both l-serine and l-OAS to form l-cystathionine. In this work, a series of site-specific variants substituting S84, Y160, and Y246 with hydrophobic residues found at the same positions in OCBSs was generated to explore the roles of the hydroxyl moieties of these residues as determinants of l-serine/l-OAS preference in TgCBS. We found that the S84A/Y160F/Y246V triple mutant behaved like an OCBS in terms of both substrate requirements, showing ß-replacement activity only with l-OAS, and pH optimum, which is decreased by ~1 pH unit. Formation of a stable aminoacrylate upon reaction with l-serine is prevented by the triple mutation, indicating the importance of the H-bonds between the hydroxyl groups of Y160, Y246, and S84 with l-serine in formation of the intermediate. Analysis of the independent effect of each mutation on TgCBS activity and investigation of the protein-aminoacrylate complex structure allowed for the conclusion that the hydroxyl group of Y246 has a major, but not exclusive, role in controlling the l-serine preference by efficiently stabilizing its leaving group. These studies demonstrate that differences in substrate specificity of CBSs are controlled by natural variations in as few as three residue positions. A better understanding of substrate specificity in TgCBS will facilitate the design of new antimicrobial compounds.

The interplay of self-assembly and target binding in centrin 1 from Toxoplasma gondii.

Centrins are conserved calcium (Ca2+)-binding proteins typically associated with centrosomes that have been implicated in several biological processes. In Toxoplasma gondii, a parasite that causes toxoplasmosis, three centrin isoforms have been recognized. We have recently characterized the metal binding and structural features of isoform 1 (TgCEN1), demonstrating that it possesses properties consistent with a role as a Ca2+ sensor and displays a Ca2+-dependent tendency to self-assemble. Herein, we expanded our studies, focusing on the self-association and target binding properties of TgCEN1 by combining biophysical techniques including dynamic light scattering, isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy. We found that the self-assembly process of TgCEN1 depends on different physicochemical factors, including Ca2+ concentration, temperature, and protein concentration, and is mediated by both electrostatic and hydrophobic interactions. The process is completely abolished upon removal of the first 21-residues of the protein and is significantly reduced in the presence of a binding target peptide derived from the human XPC protein (P17-XPC). Titration of P17-XPC to the intact protein and isolated domains showed that TgCEN1 possesses two binding sites with distinct affinities and Ca2+ sensitivity; a high-affinity site in the C-lobe which may be constitutively bound to the peptide and a low-affinity site in the N-lobe which is active only upon Ca2+ stimulus. Overall, our results suggest a specific mechanism of TgCEN1 for Ca2+-modulated target binding and support a N-to-C self-assembly mode, in which the first 21-residues of one molecule likely interact with the C-lobe of the other.

Insights into Domain Organization and Regulatory Mechanism of Cystathionine Beta-Synthase from Toxoplasma gondii.

Cystathionine beta-synthase (CBS) is a key regulator of homocysteine metabolism. Although eukaryotic CBS have a similar domain architecture with a catalytic core and a C-terminal Bateman module, their regulation varies widely across phyla. In human CBS (HsCBS), the C-terminus has an autoinhibitory effect by acting as a cap that avoids the entry of substrates into the catalytic site. The binding of the allosteric modulator AdoMet to this region alleviates this cap, allowing the protein to progress from a basal toward an activated state. The same activation is obtained by artificial removal or heat-denaturation of the Bateman module. Recently, we reported the crystal structure of CBS from Toxoplasma gondii (TgCBS) showing that the enzyme assembles into basket-like dimers similar to the basal conformers of HsCBS. These findings would suggest a similar lid function for the Bateman module which, as in HsCBS, should relax in the absence of the C-terminal module. However, herein we demonstrate that, in contrast with HsCBS, removal of the Bateman module in TgCBS through deletion mutagenesis, limited proteolysis, or thermal denaturation has no effects on its activity, oligomerization, and thermal stability. This opposite behavior we have now found in TgCBS provides evidence of a novel type of CBS regulation.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana.

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp-CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein-peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

Structural Basis for the Functional Diversity of Centrins: A Focus on Calcium Sensing Properties and Target Recognition.

Centrins are a family of small, EF hand-containing proteins that are found in all eukaryotes and are often complexed with centrosome-related structures. Since their discovery, centrins have attracted increasing interest due to their multiple, diverse cellular functions. Centrins are similar to calmodulin (CaM) in size, structure and domain organization, although in contrast to CaM, the majority of centrins possess at least one calcium (Ca2+) binding site that is non-functional, thus displaying large variance in Ca2+ sensing abilities that could support their functional versatility. In this review, we summarize current knowledge on centrins from both biophysical and structural perspectives with an emphasis on centrin-target interactions. In-depth analysis of the Ca2+ sensing properties of centrins and structures of centrins complexed with target proteins can provide useful insight into the mechanisms of the different functions of centrins and how these proteins contribute to the complexity of the Ca2+ signaling cascade. Moreover, it can help to better understand the functional redundancy of centrin isoforms and centrin-binding proteins.

Arabidopsis calmodulin-like protein CML36 is a calcium (Ca2+) sensor that interacts with the plasma membrane Ca2+-ATPase isoform ACA8 and stimulates its activity.

Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca2+)-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana We analyzed Ca2+ and Mg2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca2+/Mg2+ mixed binding sites and two low-affinity Ca2+-specific sites. Binding of Ca2+ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca2+ or Mg2+, stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca2+-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca2+ pumps.

Unique substrate specificity of ornithine aminotransferase from Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite of medical and veterinary relevance responsible for toxoplasmosis in humans. As an efficacious vaccine remains a challenge, chemotherapy is still the most effective way to combat the disease. In search of novel druggable targets, we performed a thorough characterization of the putative pyridoxal 5'-phosphate (PLP)-dependent enzyme ornithine aminotransferase from T. gondii ME49 (TgOAT). We overexpressed the protein in Escherichia coli and analysed its molecular and kinetic properties by UV-visible absorbance, fluorescence and CD spectroscopy, in addition to kinetic studies of both the steady state and pre-steady state. TgOAT is largely similar to OATs from other species regarding its general transamination mechanism and spectral properties of PLP; however, it does not show a specific ornithine aminotransferase activity like its human homologue, but exhibits both N-acetylornithine and γ-aminobutyric acid (GABA) transaminase activity in vitro, suggesting a role in both arginine and GABA metabolism in vivo The presence of Val79 in the active site of TgOAT in place of Tyr, as in its human counterpart, provides the necessary room to accommodate N-acetylornithine and GABA, resembling the active site arrangement of GABA transaminases. Moreover, mutation of Val79 to Tyr results in a change of substrate preference between GABA, N-acetylornithine and L-ornithine, suggesting a key role of Val79 in defining substrate specificity. The findings that TgOAT possesses parasite-specific structural features as well as differing substrate specificity from its human homologue make it an attractive target for anti-toxoplasmosis inhibitor design that can be exploited for chemotherapeutic intervention.

Towards Understanding Plant Calcium Signaling through Calmodulin-Like Proteins: A Biochemical and Structural Perspective.

Ca2+ ions play a key role in a wide variety of environmental responses and developmental processes in plants, and several protein families with Ca2+-binding domains have evolved to meet these needs, including calmodulin (CaM) and calmodulin-like proteins (CMLs). These proteins have no catalytic activity, but rather act as sensor relays that regulate downstream targets. While CaM is well-studied, CMLs remain poorly characterized at both the structural and functional levels, even if they are the largest class of Ca2+ sensors in plants. The major structural theme in CMLs consists of EF-hands, and variations in these domains are predicted to significantly contribute to the functional versatility of CMLs. Herein, we focus on recent advances in understanding the features of CMLs from biochemical and structural points of view. The analysis of the metal binding and structural properties of CMLs can provide valuable insight into how such a vast array of CML proteins can coexist, with no apparent functional redundancy, and how these proteins contribute to cellular signaling while maintaining properties that are distinct from CaM and other Ca2+ sensors. An overview of the principal techniques used to study the biochemical properties of these interesting Ca2+ sensors is also presented.

Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii.

Sulfur-containing amino acids play essential roles in many organisms. The protozoan parasite Toxoplasma gondii includes the genes for cystathionine ß-synthase and cystathionine γ-lyase (TgCGL), as well as for cysteine synthase, which are crucial enzymes of the transsulfuration and de novo pathways for cysteine biosynthesis, respectively. These enzymes are specifically expressed in the oocyst stage of T. gondii. However, their functionality has not been investigated. Herein, we expressed and characterized the putative CGL from T. gondii. Recombinant TgCGL almost exclusively catalyses the α,γ-hydrolysis of l-cystathionine to form l-cysteine and displays marginal reactivity toward l-cysteine. Structure-guided homology modelling revealed two striking amino acid differences between the human and parasite CGL active-sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of Asn360 to Ser demonstrated the importance of this residue in modulating the specificity for the catalysis of α,ß- versus α,γ-elimination of l-cystathionine. Replacement of Ser77 by Glu completely abolished activity towards l-cystathionine. Our results suggest that CGL is an important functional enzyme in T. gondii, likely implying that the reverse transsulfuration pathway is operative in the parasite; we also probed the roles of active-site architecture and substrate binding conformations as determinants of reaction specificity in transsulfuration enzymes.

Biochemical and biophysical characterization of a plant calmodulin: Role of the N- and C-lobes in calcium binding, conformational change, and target interaction.

In plants, transient elevation of intracellular Ca(2+) concentration in response to abiotic stress is responsible for glutamate decarboxylase (GAD) activation via association with calmodulin (CaM), an EF-hand protein consisting of two homologous domains (N and C). An unusual 1:2 binding mode of CaM to CaM-binding domains of GAD has long been known, however the contribution of the two CaM domains in target recognition and activation remains to be clarified. Here, we explored the coupling between physicochemical properties of Arabidopsis CaM1 (AtCaM1) and Arabidopsis GAD1 activation, focusing on each AtCaM1 lobe. We found that the four EF-loops of AtCaM1 differently contribute to the ~20 µM apparent affinity for Ca(2+) and the C-lobe shows a ~6-fold higher affinity than N-lobe (Kd(app) 5.6 µM and 32 µM for C- and N-lobes, respectively). AtCaM1 responds structurally to Ca(2+) in a manner similar to vertebrate CaM based on comparison of Ca(2+)-induced changes in hydrophobicity exposure, secondary structure, and hydrodynamic behavior. Molecular dynamics simulations of AtCaM1 apo and Ca(2+)-bound reveal that the latter state is significantly less flexible, although regions of the N-lobe remain quite flexible; this suggests the importance of N-lobe for completing the transition to the extended structure of holoprotein, consistent with data from ANS fluorescence, CD spectroscopy, and SEC analysis. Moreover, enzymatic analysis reveal that mutations in the two lobes affect GAD1 activation in similar ways and only intact AtCaM1 can fully activate GAD1. Taken together, our data provide new insights into the CaM lobes role in interactions between CaM and plant GAD.

Functional roles of the hexamer organization of plant glutamate decarboxylase.

Glutamate decarboxylase (GAD) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the α-decarboxylation of glutamate to γ-aminobutyrate. A unique feature of plant GAD is the presence of a calmodulin (CaM)-binding domain at its C-terminus. In plants, transient elevation of cytosolic Ca²âº in response to different types of stress is responsible for GAD activation via CaM. The crystal structure of GAD isoform 1 from Arabidopsis thaliana (AtGAD1) shows that the enzyme is a hexamer composed of a trimer of dimers. Herein, we show that in solution AtGAD1 is in a dimer-hexamer equilibrium and estimate the dissociation constant (Kd) for the hexamer under different conditions. The association of dimers into hexamers is promoted by several conditions, including high protein concentrations and low pH. Notably, binding of Ca²âº/CaM1 abolishes the dissociation of the AtGAD1 oligomer. The AtGAD1 N-terminal domain is critical for maintaining the oligomeric state as removal of the first 24 N-terminal residues dramatically affects oligomerization by producing a dimeric enzyme. The deleted mutant retains decarboxylase activity, highlighting the dimeric nature of the basic structural unit of AtGAD1. Site-directed mutagenesis identified Arg24 in the N-terminal domain as a key residue since its mutation to Ala prevents hexamer formation in solution. Both dimeric mutant enzymes form a stable hexamer in the presence of Ca²âº/CaM1. Our data clearly reveal that the oligomeric state of AtGAD1 is highly responsive to a number of experimental parameters and may have functional relevance in vivo in the light of the biphasic regulation of AtGAD1 activity by pH and Ca²âº/CaM1 in plant cells. This article is part of a special issue titled "Cofactor-Dependent Proteins: Evolution, Chemical Diversity and Bio-applications."

Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity.

It is well-established that plant hemoglobins (Hbs) are involved in nitric oxide (NO) metabolism via NO dioxygenase and/or nitrite reductase activity. The ferrous-deoxy Arabidopsis Hb1 and Hb2 (AHb1 and AHb2) have been shown to reduce nitrite to NO under hypoxia. Here, to test the hypothesis that a six- to five-coordinate heme iron transition might mediate the control of the nitrite reduction rate, we examined distal pocket mutants of AHb1 and AHb2 for nitrite reductase activity, NO production and spectroscopic features. Absorption spectra of AHbs distal histidine mutants showed that AHb1 mutant (H69L) is a stable pentacoordinate high-spin species in both ferrous and ferric states, whereas heme iron in AHb2 mutant (H66L) is hexacoordinated low-spin with Lys69 as the sixth ligand. The bimolecular rate constants for nitrite reduction to NO were 13.3 ± 0.40, 7.3 ± 0.5, 10.6 ± 0.8 and 171.90 ± 9.00 M(-1)·s(-1) for AHb1, AHb2, AHb1 H69L and AHb2 H66L, respectively, at pH 7.4 and 25 °C. Consistent with the reductase activity, the amount of NO detected by chemiluminescence was significantly higher in the AHb2 H66L mutant. Our data indicate that nitrite reductase activity is determined not only by heme coordination, but also by a unique distal heme pocket in each AHb.

Role of active-site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C-S lyase.

In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C-S lyase from Corynebacterium diphtheriae is a pyridoxal 5'-phosphate-dependent enzyme in the transsulfuration pathway that catalyzes the α,ß-elimination of sulfur-containing amino acids, such as L-cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L-methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active-site residues, Y55F and Y114F, were characterized by UV-visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady-state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130-fold decrease in K(d)(PLP) at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP-binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of L-cystathionine, and causes a change in substrate specificity towards L-serine and O-acetyl-L-serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C-S lyase, which seems to be regulated by active-site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors.

Catalytic specificity and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa.

The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H2S) via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa (PaCGL), a multidrug-opportunistic pathogen chiefly responsible for nosocomial infections. In addition to a canonical L-cystathionine hydrolysis, PaCGL efficiently catalyzes the production of H2S using L-cysteine and/or L-homocysteine as alternative substrates. Comparative analysis with the human enzyme and counterparts from other pathogens revealed distinct structural features within the primary enzyme cavities. Specifically, a distinctly folded entrance loop could potentially modulate the access of substrates and/or inhibitors to the catalytic site. Our findings offer significant insights into the structural evolution of CGL enzymes across different pathogens and provide novel opportunities for developing specific inhibitors targeting PaCGL.

Structural Basis for Cyclosporin Isoform-Specific Inhibition of Cyclophilins from Toxoplasma gondii.

Cyclosporin (CsA) has antiparasite activity against the human pathogen Toxoplasma gondii. A possible mechanism of action involves CsA binding to T. gondii cyclophilins, although much remains to be understood. Herein, we characterize the functional and structural properties of a conserved (TgCyp23) and a more divergent (TgCyp18.4) cyclophilin isoform from T. gondii. While TgCyp23 is a highly active cis-trans-prolyl isomerase (PPIase) and binds CsA with nanomolar affinity, TgCyp18.4 shows low PPIase activity and is significantly less sensitive to CsA inhibition. The crystal structure of the TgCyp23:CsA complex was solved at the atomic resolution showing the molecular details of CsA recognition by the protein. Computational and structural studies revealed relevant differences at the CsA-binding site between TgCyp18.4 and TgCyp23, suggesting that the two cyclophilins might have distinct functions in the parasite. These studies highlight the extensive diversification of TgCyps and pave the way for antiparasite interventions based on selective targeting of cyclophilins.

Role of myristoylation in modulating PCaP1 interaction with calmodulin.

Plasma membrane-associated Cation-binding Protein 1 (PCaP1) belongs to the plant-unique DREPP protein family with largely unknown biological functions but ascertained roles in plant development and calcium (Ca2+) signaling. PCaP1 is anchored to the plasma membrane via N-myristoylation and a polybasic cluster, and its N-terminal region can bind Ca2+/calmodulin (CaM). However, the molecular determinants of PCaP1-Ca2+-CaM interaction and the functional impact of myristoylation in the complex formation and Ca2+ sensitivity of CaM remained to be elucidated. Herein, we investigated the direct interaction between Arabidopsis PCaP1 (AtPCaP1) and CaM1 (AtCaM1) using both myristoylated and non-myristoylated peptides corresponding to the N-terminal region of AtPCaP1. ITC analysis showed that AtCaM1 forms a high affinity 1:1 complex with AtPCaP1 peptides and the interaction is strictly Ca2+-dependent. Spectroscopic and kinetic Ca2+ binding studies showed that the myristoylated peptide dramatically increased the Ca2+-binding affinity of AtCaM1 and slowed the Ca2+ dissociation rates from both the C- and N-lobes, thus suggesting that the myristoylation modulates the mechanism of AtPCaP1 recognition by AtCaM1. Furthermore, NMR and CD spectroscopy revealed that the structure of both the N- and C-lobes of Ca2+-AtCaM1 changes markedly in the presence of the myristoylated AtPCaP1 peptide, which assumes a helical structure in the final complex. Overall, our results indicate that AtPCaP1 biological function is strictly related to the presence of multiple ligands, i.e., the myristoyl moiety, Ca2+ ions and AtCaM1 and only a full characterization of their equilibria will allow for a complete molecular understanding of the putative role of PCaP1 as signal protein.

Conformational Plasticity of Centrin 1 from Toxoplasma gondii in Binding to the Centrosomal Protein SFI1.

Centrins are calcium (Ca2+)-binding proteins that are involved in many cellular functions including centrosome regulation. A known cellular target of centrins is SFI1, a large centrosomal protein containing multiple repeats that represent centrin-binding motifs. Recently, a protein homologous to yeast and mammalian SFI1, denominated TgSFI1, which shares SFI1-repeat organization, was shown to colocalize at centrosomes with centrin 1 from Toxoplasma gondii (TgCEN1). However, the molecular details of the interaction between TgCEN1 and TgSFI1 remain largely unknown. Herein, combining different biophysical methods, including isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy, we determined the binding properties of TgCEN1 and its individual N- and C-terminal domains to synthetic peptides derived from distinct repeats of TgSFI1. Overall, our data indicate that the repeats in TgSFI1 constitute binding sites for TgCEN1, but the binding modes of TgCEN1 to the repeats differ appreciably in terms of binding affinity, Ca2+ sensitivity, and lobe-specific interaction. These results suggest that TgCEN1 displays remarkable conformational plasticity, allowing for the distinct repeats in TgSFI1 to possess precise modes of TgCEN1 binding and regulation during Ca2+ sensing, which appears to be crucial for the dynamic association of TgCEN1 with TgSFI1 in the centrosome architecture.

Oxygen binding to Arabidopsis thaliana AHb2 nonsymbiotic hemoglobin: evidence for a role in oxygen transport.

Nonsymbiotic hemoglobins AHb1 and AHb2 discovered in Arabidopsis thaliana are likely to carry out distinct physiological roles, in consideration of their differences in sequence, structure, expression pattern, and tissue localization. Despite a relatively fast autoxidation in the presence of O(2) , we were able to collect O(2) -binding curves for AHb2 in the presence of a reduction enzymatic system. AHb2 binds O(2) noncooperatively with a p50 of 0.021 ± 0.003 Torr, a value consistent with a recently proposed role in O(2) transport. The analysis of the internal cavities derived from the structures sampled in molecular dynamics simulations confirms strong differences with AHb1, proposed to work as a NO deoxygenase in vivo. Overall, our results are consistent with a role for AHb2 as an oxygen carrier, as recently proposed on the basis of experiments on AHb2-overexpressing mutants of A. thaliana.

Oxygen and nitric oxide rebinding kinetics in nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana.

Type 1 nonsymbiotic hemoglobin from Arabidopsis thaliana (AHb1) shows a partial bis-histidyl hexacoordination but can reversibly bind diatomic ligands. The physiological function is still unclear, but the high oxygen affinity rules out a function related to O2 sensing, carrying, or storing. To gain insight into its possible functional roles, we have investigated its O2 and NO rebinding kinetics after laser flash photolysis. The rate constants of the rebinding from the primary docking site for both O2 and NO are higher than CO, with lower photolysis yields. Moreover, the amplitude of the geminate phase increases and, as for CO, the numerical analysis of the experimental curves suggests the existence of an internal pathway leading, with high rate, to an additional docking site. However, the accessibility to this site seems to be strongly ligand-dependent, being lower for O2 and higher for NO.

Structural insight into the unique conformation of cystathionine ß-synthase from Toxoplasma gondii.

Cysteine plays a major role in the redox homeostasis and antioxidative defense mechanisms of many parasites of the phylum Apicomplexa. Of relevance to human health is Toxoplasma gondii, the causative agent of toxoplasmosis. A major route of cysteine biosynthesis in this parasite is the reverse transsulfuration pathway involving two key enzymes cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGL). CBS from T. gondii (TgCBS) catalyzes the pyridoxal-5́-phosphate-dependent condensation of homocysteine with either serine or O-acetylserine to produce cystathionine. The enzyme can perform alternative reactions that use homocysteine and cysteine as substrates leading to the endogenous biosynthesis of hydrogen sulfide, another key element in maintaining the intracellular redox equilibrium. In contrast with human CBS, TgCBS lacks the N-terminal heme binding domain and is not responsive to S-adenosylmethionine. Herein, we describe the structure of a TgCBS construct that lacks amino acid residues 466-491 and shows the same activity of the native protein. TgCBS Δ466-491 was determined alone and in complex with reaction intermediates. A complementary molecular dynamics analysis revealed a unique domain organization, similar to the pathogenic mutant D444N of human CBS. Our data provides one missing piece in the structural diversity of CBSs by revealing the so far unknown three-dimensional arrangement of the CBS-type of Apicomplexa. This domain distribution is also detected in yeast and bacteria like Pseudomonas aeruginosa. These results pave the way for understanding the mechanisms by which TgCBS regulates the intracellular redox of the parasite, and have far-reaching consequences for the functional understanding of CBSs with similar domain distribution.