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 β-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,β-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

Characterization of putative PLP-dependent beta C-S lyase from Corynebacterium diphtheriae, a possible target for a new antimicrobial agent.

The continuous emergence of antibiotic resistance in microbial pathogens requires a sustained effort to identify new antimicrobial compounds and targets. The biosynthesis of methionine is an attractive target given its importance in protein and DNA metabolism. Moreover, most of the steps in this pathway are absent in mammals, lessening the opportunity of unwanted side effects. Herein, detailed biochemical characterization of a putative pyridoxal 5\u2019-phosphate (PLP)-dependent beta C-S lyase from Corynebacterium diphtheriae, a pathogenic bacterium that causes diphtheria, has been performed. We overexpressed the protein in Escherichia coli and analyzed substrate specificity, pH dependence of steady state kinetic parameters and ligand-induced spectral transitions of the recombinant protein by a combination of UV/Vis and fluorescence spectroscopy. The 3D structure of beta C-S lyase from Corynebacterium diphtheriae has already been solved at 1.99 \uc5 resolution (Joint Center for Structural Genomics). The enzyme is a homodimer composed of ~42 kDa subunits, each associated with one molecule of PLP. Structural comparison of beta C-S lyase from Corynebacterium diphtheriae with beta C-S lyase from Streptococcus anginosus1 and cystalysin from Treponema denticola2 indicates a similarity in overall folding and active site residues. We used site-directed mutagenesis to highlight the importance of the active site residues Tyr55, Tyr114, and Arg351, analyzing the effects of amino acid replacement on catalytic properties and spectra of enzyme-ligand complexes. Better understanding of the active site of Corynebacterium diphtheriae beta C-S lyase and the determinants of substrate and reaction specificity from this work will facilitate the design of novel inhibitors, as antibacterial therapeutics

Functional Roles of the Hexamer Organization of Plant Glutamate Decarboxylase

Glutamate decarboxylase (GAD) is a pyridoxal 5\ub4-phosphate (PLP)-dependent enzyme that catalyzes the irreversible \u3b1-decarboxylation of glutamate to \u3b3-aminobutyrate. The enzyme is widely distributed in eukaryotes and prokaryotes, although its function varies in different organisms. 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 Ca2+ in response to different types of stress is thought to be responsible for GAD activation via CaM. The crystal structure of GAD1 from Arabidopsis thaliana shows that the enzyme is a hexamer composed of trimer-of-dimers. Herein, we show that in solution GAD1 exists as a dimer/hexamer equilibrium mixture, and we estimate the dissociation constant (Kd) for the hexamer under different conditions. The association of dimers into hexamers is promoted by a number of conditions, including high protein concentrations and low pH. Notably, binding of Ca2+/CaM abolishes GAD1 oligomer dissociation by forming a stable complex in which three CaM bind to a GAD1 hexamer. The GAD1 N-terminal domain is critical for maintaining the oligomeric state, since the removal of the first 24 N-terminal residues dramatically affects the oligomerization process by producing an enzyme that exists only as a dimer. The deleted mutant retains decarboxylase activity, highlighting the dimeric nature of the basic structural unit of GAD1. Site-directed mutagenesis identified a hexamerization \u2018hot spot\u2019 centered on Arg24 in the N-terminal domain. Mutation of this critical Arg residue to Ala prevents hexamer formation in solution. Surprisingly, both the dimeric Arg\uf0e0Ala and \uf0441-24 mutant enzymes form a stable hexamer in the presence of Ca2+/CaM. The present data, clearly revealing that the GAD1 oligomeric state is highly responsive to a number of experimental parameters, might have functional relevance in vivo and is discussed in the light of the biphasic regulation of GAD1 activity by pH and Ca2+/CaM in plant cells

Functional Characterization of a new Lipid Transfer Protein With Antimicrobial Properties From Medicago truncatula

Non specific lipid transfer proteins form a multigenic protein family in plants. They are able to bind in vitro different lipids but their function in vivo remains speculative. They have been suggested to participate to many aspects of plant physiology and cell biology, including assembly of extracellular hydrophobic polymers and involvement in stress and pathogen responses. To understand whether MtN5, a new LTP2 identified in Medicago truncatula roots, plays a possible role in the plant protection function, we investigated its antifungal and antibacterial properties. A cDNA sequence encoding the protein, without the signal peptide, was cloned in a prokaryotic expression system. The purified recombinant protein has been characterized for its lipid binding properties and for its in vitro antimicrobial properties. The results showed that the recombinant MtN5 is able to bind lipids, similarly to other members of the LTP protein family and possesses a selective action against specific pathogens, potentially due to interactions with microbe specific membrane structures

Functional characterization of a new Lipid Transfer Protein with antimicrobial properties from Medicago truncatula

Non specific Lipid Transfer Proteins (nsLTPs) form a multigenic protein family in plants. They are able to bind in vitro different lipids but their function in vivo remains speculative. They have been suggested to participate to many aspects of plant physiology and cell biology, including assembly of extracellular hydrophobic polymers and involvement in stress and pathogen responses. To understand whether MtN5, a new LTP2 identified in Medicago truncatula nodulated roots, plays a possible role in plant defence mechanisms, we investigated its antifungal and antibacterial properties

Nanodevice-induced conformational and functional changes in a prototypical calcium sensor protein.

Calcium (Ca2+) plays a major role in a variety of cellular processes. Fine changes in its concentration are detected by calcium sensor proteins, which adopt specific conformations to regulate their molecular targets. Here, two distinct nanodevices were probed as biocompatible carriers of Ca2+-sensors and the structural and functional effects of protein-nanodevice interactions were investigated. The prototypical Ca2+-sensor recoverin (Rec) was incubated with 20-25 nm CaF2 nanoparticles (NPs) and 70-80 nm liposomes with lipid composition similar to that found in photoreceptor cells. Circular dichroism and fluorescence spectroscopy were used to characterize changes in the protein secondary and tertiary structure and in thermal stability upon interaction with the nanodevice, both in the presence and in the absence of free Ca2+. Variations in the hydrodynamic diameter of the complex were measured by dynamic light scattering and the residual capability of the protein to act as a Ca2+-sensor in the presence of NPs was estimated spectroscopically. The conformation, thermal stability and Ca2+-sensing capability of Rec were all significantly affected by the presence of NPs, while liposomes did not significantly perturb Rec conformation and function, allowing reversible binding. NP-bound Rec maintained an all-helical fold but showed lower thermal stability and high cooperativity of unfolding. Our analysis can be proficiently used to validate the biocompatibility of other nanodevices intended for biomedical applications involving Ca2+-sensors

Studies on the role played by MtN5, a root-specific LTP from Medicago truncatula expressed during rhizobia infection

Non specific lipid transfer proteins (nsLTPs) form a multigenic protein family in plants. These proteins are characterized by their capacity to in vitro bind different lipids, such as fatty acids and phospholipids. Their functions in vivo are still unclear, even if it has been hypothesized a role for LTP in the assembly of extra cellular hydrophobic polymers (e.g. cutin and suberin), in signalling and in plant defence against pathogens. A root specific nsLTP mRNA (MtN5) has been identified in Medicago truncatula and it has been reported to be expressed during the early phases of nodulation. It has been proposed that MtN5 could be involved either in a general defence mechanism against rhizobia or in Nod factor signalling. In this work we have studied the expression of MtN5 during the nodulation process induced by Sinorhizobium meliloti 1021 wild-type and by an engineered S. meliloti with enhanced capacity to nodulate M. truncatula. Furthermore, MtN5 (Y15371) cDNA has been cloned and expressed in a prokaryotic system (E. coli BL21 DE3 pLysS) in order to study its properties in vitro

Ligand Migration and Binding in Nonsymbiotic Hemoglobins of Arabidopsis thaliana

We have studied carbon monoxide (CO) migration and binding in the nonsymbiotic hemoglobins AHb1 and AHb2 of Arabidopsis thaliana using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures. Both proteins have similar amino acid sequences but display pronounced differences in ligand binding properties, at both physiological and cryogenic temperatures. Near neutral pH, the distal HisE7 side chain is close to the heme-bound ligand in the majority of AHb1-CO molecules, as indicated by a low CO stretching frequency at 1921 cm(-1). In this fraction, two CO docking sites can be populated, the primary site B and the secondary site C. When the pH is lowered, a high-frequency stretching band at approximately 1964 cm(-1) grows at the expense of the low-frequency band, indicating that HisE7 protonates and, concomitantly, moves away from the bound ligand. Geminate rebinding barriers are markedly different for the two conformations, and docking site C is not accessible in the low-pH conformation. Rebinding of NO ligands was observed only from site B of AHb1, regardless of conformation. In AHb2, the HisE7 side chain is removed from the bound ligand; rebinding barriers are low, and CO molecules can populate only primary docking site B. These results are interpreted in terms of differences in the active site structures and physiological functions

Rapid kinetics of calcium dissociation from plant calmodulin and calmodulin-like proteins and effect of target peptides

Calcium (Ca2+) signaling represents a universal information code in plants, playing crucial roles spanning developmental processes to stress responses. Ca2+ signals are decoded into defined plant adaptive responses by different Ca2+ sensing proteins, including calmodulin (CaM) and calmodulin-like (CML) proteins. Although major advances have been achieved in describing how these Ca2+ decoding proteins interact and regulate downstream target effectors, the molecular details of these processes remain largely unknown. Herein, the kinetics of Ca2+ dissociation from a conserved CaM and two CML isoforms from A. thaliana has been studied by fluorescence stopped-flow spectroscopy. Kinetic data were obtained for the isolated Ca2+-bound proteins as well as for the proteins complexed with different target peptides. Moreover, the lobe specific interactions between the Ca2+ sensing proteins and their targets were characterized by using a panel of protein mutants deficient in Ca2+ binding at the N-lobe or C-lobe. Results were analyzed and discussed in the context of the Ca2+-decoding and Ca2+-controlled target binding mechanisms in plants

Ligand Migration and Binding in Non-Symbiotic Hemoglobins of Arabidopsis thaliana

We have studied carbon monoxide (CO) migration and binding in the nonsymbiotic hemoglobins AHb1 and AHb2 of Arabidopsis thaliana using Fourier transform infrared (FTIR) spectroscopy combined with temperature derivative spectroscopy (TDS) at cryogenic temperatures. Both proteins have similar amino acid sequences but display pronounced differences in ligand binding properties, at both physiological and cryogenic temperatures. Near neutral pH, the distal HisE7 side chain is close to the heme-bound ligand in the majority of AHb1-CO molecules, as indicated by a low CO stretching frequency at 1921 cm−1. In this fraction, two CO docking sites can be populated, the primary site B and the secondary site C. When the pH is lowered, a high-frequency stretching band at 1964 cm−1 grows at the expense of the low-frequency band, indicating that HisE7 protonates and, concomitantly, moves away from the bound ligand. Geminate rebinding barriers are markedly different for the two conformations, and docking site C is not accessible in the low-pH conformation. Rebinding of NO ligands was observed only from site B of AHb1, regardless of conformation. In AHb2, the HisE7 side chain is removed from the bound ligand; rebinding barriers are low, and CO molecules can populate only primary docking site B. These results are interpreted in terms of differences in the active site structures and physiological functions