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Heat shock proteins (HSPs), especially Hsp70 (HSPA1), have been associated with cellular protection from various cellular stresses including heat, hypoxia-ischemia, neurodegeneration, toxins, and trauma. Endogenous HSPs are often synthesized in direct response to these stresses but in many situations are inadequate in protecting cells. The present study addresses the transduction of Hsp70 into cells providing protection from acute oxidative stress by H2O2. The recombinant Fv-Hsp70 protein and two mutant Fv-Hsp70 proteins minus the ATPase domain and minus the ATPase and terminal lid domains were tested at 0.5 and 1.0 μM concentrations after two different concentrations of H2O2 treatment. All three recombinant proteins protected SH-SY5Y cells from acute H2O2 toxicity. This data indicated that the protein binding domain was responsible for cellular protection. In addition, experiments pretreating cells with inhibitors of antioxidant proteins catalase and gamma-glutamylcysteine synthase (GGCS) before H2O2 resulted in cell death despite treatment with Fv-Hsp70, implying that both enzymes were protected from acute oxidative stress after treatment with Fv-Hsp70. This study demonstrates that Fv-Hsp70 is protective in our experiments primarily by the protein-binding domain. The Hsp70 terminal lid domain was also not necessary for protection.

Chagas disease is a neglected tropical disease (NTD) caused by Trypanosoma cruzi, whilst leishmaniasis, which is caused by over 20 species of Leishmania, represents a group of NTDs endemic to most countries in the tropical and subtropical belt of the planet. These diseases remain a significant health problem both in endemic countries and globally. These parasites and other trypanosomatids, including T. theileri, a bovine pathogen, rely on cysteine biosynthesis for the production of trypanothione, which is essential for parasite survival in hosts. The de novo pathway of cysteine biosynthesis requires the conversion of O‐acetyl‐l‐serine into l‐cysteine, which is catalysed by cysteine synthase (CS). These enzymes present potential for drug development against T. cruzi, Leishmania spp. and T. theileri. To enable these possibilities, biochemical and crystallographic studies of CS from T. cruzi (TcCS), L. infantum (LiCS) and T. theileri (TthCS) were conducted. Crystal structures of the three enzymes were determined at resolutions of 1.80 Å for TcCS, 1.75 Å for LiCS and 2.75 Å for TthCS. These three homodimeric structures show the same overall fold and demonstrate that the active‐site geometry is conserved, supporting a common reaction mechanism. Detailed structural analysis revealed reaction intermediates of the de novo pathway ranging from an apo structure of LiCS and holo structures of both TcCS and TthCS to the substrate‐bound structure of TcCS. These structures will allow exploration of the active site for the design of novel inhibitors. Additionally, unexpected binding sites discovered at the dimer interface represent new potential for the development of protein–protein inhibitors. [ABSTRACT FROM AUTHOR]

The present study analyzed the effects of individual microbes and their consortium (Priestia endophytica NDAS01F , Bacillus licheniformis NDSA24R, and P. flexa NDAS28R) either alone or in interaction with thiourea (TU) on growth and responses of rice plants subjected to As stress (50 mg kg−1 in soil) in a pot experiment. The bacteria used in the experiment were isolated from As contaminated fields of Nadia, West Bengal and showed significant As removal potential in in vitro experiment. The results revealed significant growth improvement, biomass accumulation, and decline in malondialdehyde levels in rice plants in bacterial and TU treatments as compared to control As treatment. The best results were observed in a bacterial consortium (B1-2-3), which induced a profound increase of 65%, 43%, 127% and 83% in root length, shoot length, leaf width and fresh weight, respectively. Sulfur metabolism and cell wall synthesis were stimulated upon bacterial and TU amendment in plants. The maximum reduction in As concentration was observed in B2 in roots (−55%) and in B1-2-3 in shoot (−83%). The combined treatment of B1-2-3 + TU proved to be less effective as compared to that of B1-2-3 in terms of As reduction and growth improvement. Hence, the usage of bacterial consortium obtained in the present work is a sustainable approach, which might find relevance in field conditions to achieve As reduction in rice grains and to attain higher growth of plants without the need for additional TU supplementation. [Display omitted] • Bacteria isolated from Nadia, West Bengal were used for consortium. • Bacterial consortium promoted growth of plants and reduced arsenic concentration. • Arsenic translocation from root to shoot was significantly decreased. • Bacterial consortium + thiourea did not result in synergistic effects on growth. • Bacterial consortium + thiourea did not cause additional arsenic reduction. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is a highly successful enteric pathogen with a small, host-adapted genome (1.64 Mbp, ~1650 coding genes). As a result, C. jejuni has limited capacity in numerous metabolic pathways, including sulfur metabolism. Unable to utilise ionic sulfur, C. jejuni relies on the uptake of exogenous cysteine and its derivatives for its supply of this essential amino acid. Cysteine can also be synthesized de novo by the sole cysteine synthase, CysM. In this study, we explored the substrate specificity of purified C. jejuni CysM and define it as an O-acetyl-L-serine sulfhydrylase with an almost absolute preference for sulfide as sulfur donor. Sulfide is produced in abundance in the intestinal niche C. jejuni colonises, yet sulfide is generally viewed as highly toxic to bacteria. We conducted a series of growth experiments in sulfur-limited media and demonstrate that sulfide is an excellent sulfur source for C. jejuni at physiologically relevant concentrations, combating the view of sulfide as a purely deleterious compound to bacteria. Nonetheless, C. jejuni is indeed inhibited by elevated concentrations of sulfide and we sought to understand the targets involved. Surprisingly, we found that inactivation of the sulfide-sensitive primary terminal oxidase, the cbb3-type cytochrome c oxidase CcoNOPQ, did not explain the majority of growth inhibition by sulfide. Therefore, further work is required to reveal the cellular targets responsible for sulfide toxicity in C. jejuni. [ABSTRACT FROM AUTHOR]

Background: Chagas disease is a potentially fatal disease caused by the parasite Trypanosoma cruzi. There is growing scientific interest in finding new and better therapeutic alternatives for this disease’s treatment. Methods: A total of 81 terpene compounds with potential trypanocidal activity were screened and found to have potential T. cruzi cysteine synthase (TcCS) inhibition using molecular docking, molecular dynamics, ADME and PAIN property analyses and in vitro susceptibility assays. Results: Molecular docking analyses revealed energy ranges from −10.5 to −4.9 kcal/mol in the 81 tested compounds, where pentacyclic triterpenes were the best. Six compounds were selected to assess the stability of the TcCS–ligand complexes, of which lupeol acetate (ACLUPE) and α-amyrin (AMIR) exhibited the highest stability during 200 ns of molecular dynamics analysis. Such stability was primarily due to their hydrophobic interactions with the amino acids located in the enzyme’s active site. In addition, ACLUPE and AMIR exhibited lipophilic characteristics, low intestinal absorption and no structural interferences or toxicity. Finally, selective index for ACLUPE was >5.94, with moderate potency in the trypomastigote stage (EC50 = 15.82 ± 3.7 μg/mL). AMIR’s selective index was >9.36 and it was moderately potent in the amastigote stage (IC50 = 9.08 ± 23.85 μg/mL). Conclusions: The present study proposes a rational approach for exploring lupeol acetate and α-amyrin terpene compounds to design new drugs candidates for Chagas disease.

Contact-dependent growth inhibition (CDI) is a wide-spread mechanism of inter-bacterial competition. CDI+ bacteria deliver CdiA-CT toxins into neighboring bacteria and produce specific immunity proteins that protect against self-intoxication. The CdiA-CT toxin from uropathogenic Escherichia coli 536 is a latent tRNase that is only active when bound to the cysteine biosynthetic enzyme CysK. Remarkably, the CysK:CdiA-CT binding interaction mimics the 'cysteine synthase' complex of CysK:CysE. The C-terminal tails of CysE and CdiA-CT each insert into the CysK active-site cleft to anchor the respective complexes. The dissociation constant for CysK:CdiA-CT (K d ~ 11 nM) is comparable to that of the E. coli cysteine synthase complex (K d ~ 6 nM), and both complexes bind through a two-step mechanism with a slow isomerization phase after the initial encounter. However, the second-order rate constant for CysK:CdiA-CT binding is two orders of magnitude slower than that of the cysteine synthase complex, suggesting that CysE should outcompete the toxin for CysK occupancy. However, we find that CdiA-CT can effectively displace CysE from pre-formed cysteine synthase complexes, enabling toxin activation even in the presence of excess competing CysE. This adventitious binding, coupled with the very slow rate of CysK:CdiA-CT dissociation, ensures robust nuclease activity in target bacteria.

Owing to the global health crisis of resistant pathogenic infections, researchers are emphasizing the importance of novel prevention and control strategies. Existing antimicrobial drugs predominantly target a few pathways, and their widespread use has pervasively increased drug resistance. Therefore, it is imperative to develop new antimicrobial drugs with novel targets and chemical structures. The de novo cysteine biosynthesis pathway, one of the microbial metabolic pathways, plays a crucial role in pathogenicity and drug resistance. This pathway notably differs from that in humans, thereby representing an unexplored target for developing antimicrobial drugs. Herein, we have presented an overview of cysteine biosynthesis pathways and their roles in the pathogenicity of various microorganisms. Additionally, we have investigated the structure and function of enzymes involved in these pathways as well as have discussed drug design strategies and structure–activity relationships of the enzyme inhibitors. This review provides valuable insights for developing novel antimicrobials and offers new avenues to combat drug resistance. [Display omitted] • Cysteine biosynthesis pathway plays key role in the pathogenicity and drug resistance of pathogens. • Structures and functions of key enzymes in cysteine biosynthesis. • Drug design strategies and the structure–activity relationships of inhibitors targeting cysteine biosynthesis. [ABSTRACT FROM AUTHOR]

Contact-dependent growth inhibition (CDI) is a widespread mechanism of bacterial competition. CDI(+) bacteria deliver the toxic C-terminal region of contact-dependent inhibition A proteins (CdiA-CT) into neighboring target bacteria and produce CDI immunity proteins (CdiI) to protect against self-inhibition. The CdiA-CT(EC536) deployed by uropathogenic Escherichia coli 536 (EC536) is a bacterial toxin 28 (Ntox28) domain that only exhibits ribonuclease activity when bound to the cysteine biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK). Here, we present crystal structures of the CysK/CdiA-CT(EC536) binary complex and the neutralized ternary complex of CysK/CdiA-CT/CdiI(EC536) CdiA-CT(EC536) inserts its C-terminal Gly-Tyr-Gly-Ile peptide tail into the active-site cleft of CysK to anchor the interaction. Remarkably, E. coli serine O-acetyltransferase uses a similar Gly-Asp-Gly-Ile motif to form the "cysteine synthase" complex with CysK. The cysteine synthase complex is found throughout bacteria, protozoa, and plants, indicating that CdiA-CT(EC536) exploits a highly conserved protein-protein interaction to promote its toxicity. CysK significantly increases CdiA-CT(EC536) thermostability and is required for toxin interaction with tRNA substrates. These observations suggest that CysK stabilizes the toxin fold, thereby organizing the nuclease active site for substrate recognition and catalysis. By contrast, Ntox28 domains from Gram-positive bacteria lack C-terminal Gly-Tyr-Gly-Ile motifs, suggesting that they do not interact with CysK. We show that the Ntox28 domain from Ruminococcus lactaris is significantly more thermostable than CdiA-CT(EC536), and its intrinsic tRNA-binding properties support CysK-independent nuclease activity. The striking differences between related Ntox28 domains suggest that CDI toxins may be under evolutionary pressure to maintain low global stability.

We show that Mycobacterium smegmatis mutants disrupted in mscR, coding for a dual function S-nitrosomycothiol reductase and formaldehyde dehydrogenase, and mshC, coding for a mycothiol ligase and lacking mycothiol (MSH), are more susceptible to S-nitrosoglutathione (GSNO) and aldehydes than wild type. MSH is a cofactor for MscR, and both mshC and mscR are induced by GSNO and aldehydes. We also show that a mutant disrupted in egtA, coding for a γ-glutamyl cysteine synthetase and lacking in ergothioneine, is sensitive to nitrosative stress but not to aldehydes. In addition, we find that MSH and S-nitrosomycothiol reductase are required for normal biofilm formation in M. smegmatis, suggesting potential new therapeutic pathways to target to inhibit or disrupt biofilm formation. © 2016 IUBMB Life, 68(8):621-628, 2016.

Campylobacter jejuni is a highly successful enteric pathogen with a small, host-adapted genome (1.64 Mbp, ~1650 coding genes). As a result, C. jejuni has limited capacity in numerous metabolic pathways, including sulfur metabolism. Unable to utilise ionic sulfur, C. jejuni relies on the uptake of exogenous cysteine and its derivatives for its supply of this essential amino acid. Cysteine can also be synthesized de novo by the sole cysteine synthase, CysM. In this study, we explored the substrate specificity of purified C. jejuni CysM and define it as an O-acetyl-L-serine sulfhydrylase with an almost absolute preference for sulfide as sulfur donor. Sulfide is produced in abundance in the intestinal niche C. jejuni colonises, yet sulfide is generally viewed as highly toxic to bacteria. We conducted a series of growth experiments in sulfur-limited media and demonstrate that sulfide is an excellent sulfur source for C. jejuni at physiologically relevant concentrations, combating the view of sulfide as a purely deleterious compound to bacteria. Nonetheless, C. jejuni is indeed inhibited by elevated concentrations of sulfide and we sought to understand the targets involved. Surprisingly, we found that inactivation of the sulfide-sensitive primary terminal oxidase, the cbb3-type cytochrome c oxidase CcoNOPQ, did not explain the majority of growth inhibition by sulfide. Therefore, further work is required to reveal the cellular targets responsible for sulfide toxicity in C. jejuni.

Haemonchus contortus , commonly known as Barber's pole worm, is an economically important gastrointestinal nematode of sheep and goats especially in tropical and sub-tropical regions of the world. Cysteine synthesis is a very important metabolic pathway for the parasite, however the functional aspects of cysteine synthesis in parasite are largely unknown. The key question which we have investigated in the study is; whether the parasite uses a de novo pathway of cysteine synthesis, which is unknown in multicellular organisms of the animal kingdom and known to be absent in mammals. Directional cloning of the cysteine synthase (CS) gene was done in pET303 champion vector using restriction sites XbaI and XhoI. The CS gene of the H.contortus was closely related to CS-A protein of Oesophagostomum dentatum and a hypothetical protein of Ancylostoma ceylanicum. Recombinant protein of the H contortus CS (rHC-CS) gene was expressed using pET303 vector in pLysS BL21 strain of E.coli and subsequently purified by Ni-NTA affinity chromatography. Western blot using anti-His tag antibody confirmed the presence of rHC-CS. Biochemical assay, FTIR and enzyme kinetics studies revealed that rHC-CS used O-acetyl serine as substrate to produce cysteine using de novo pathway and CS activity was also confirmed with the homogenate of H.contortus. Upregulation of CS transcripts in the adult and its downregulation in the L3 larval stage suggests that de novo pathway contributes to the cysteine requirement of mature H.contortus. It is concluded that de novo pathway is an active metabolic pathway in H.contortus. [Display omitted] • Elucidation of de novo pathway of cysteine synthesis in H.contortus. • Molecular characterization and functional expression of the cysteine synthase protein of H.contortus. • Mapping of expression of CS gene at different stages of life cycle of parasite. [ABSTRACT FROM AUTHOR]

O-acetylserine sulfhydrylase A (CysK) is the pyridoxal 5'-phosphate-dependent enzyme that catalyzes the final reaction of cysteine biosynthesis in bacteria. CysK was initially identified in a complex with serine acetyltransferase (CysE), which catalyzes the penultimate reaction in the synthetic pathway. This "cysteine synthase" complex is stabilized by insertion of the CysE C-terminus into the active-site of CysK. Remarkably, the CysK/CysE binding interaction is conserved in most bacterial and plant systems. For the past 40years, CysK was thought to function exclusively in cysteine biosynthesis, but recent studies have revealed a repertoire of additional "moonlighting" activities for this enzyme. CysK and its paralogs influence transcription in both Gram-positive bacteria and the nematode Caenorhabditis elegans. CysK also activates an antibacterial nuclease toxin produced by uropathogenic Escherichia coli. Intriguingly, each moonlighting activity requires a binding partner that invariably mimics the C-terminus of CysE to interact with the CysK active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

Plants catalyze the biosynthesis of a large number of non-protein amino acids, which are usually toxic for other organisms. In this review, the chemistry and metabolism of N–heterocyclic non-protein amino acids from plants are described. These N–heterocyclic non-protein amino acids are composed of β-substituted alanines and include mimosine, β–pyrazol–1–yl–l–alanine, willardiine, isowillardiine, and lathyrine. These β-substituted alanines consisted of an N–heterocyclic moiety and an alanyl side chain. This review explains how these individual moieties are derived from their precursors and how they are used as the substrate for biosynthesizing the respective N–heterocyclic non-protein amino acids. In addition, known catabolism and possible role of these non-protein amino acids in the actual host is explained. [ABSTRACT FROM AUTHOR]

Pyridoxal-5′-phosphate-dependent cysteine synthases synthesize l -cysteine from their primary substrates, O -acetyl- l -serine (OAS) and O -phospho- l -serine (OPS), and their secondary substrate, sulfide. The mechanism by which cysteine synthases recognize OPS remains unclear; hence, we investigated the OPS recognition mechanism of the OPS sulfhydrylase obtained from Aeropyrum pernix K1 (ApOPSS) and the OAS sulfhydrylase-B obtained from Escherichia coli (EcOASS-B), using protein engineering methods. From the amino acid sequence alignment data, we found that some OPS sulfhydrylases (OPSSs) had a Tyr corresponding to the Phe225 and Phe141 residues in ApOPSS and EcOASS-B, respectively, and that the Tyr residue could facilitate OPS recognition. The enzymatic activity of the ApOPSS F225Y mutant toward OPS decreased compared with that of the wild-type; the k cat value decreased 2.3-fold during cysteine synthesis. X-ray crystallography results of the complex of ApOPSS F225Y and F225Y/R297A mutants bound to OPS and l -cysteine showed that k cat might have decreased because of the stronger interactions of the reaction product phosphate with Tyr225, Thr203, and Arg297, and that of the l -cysteine with Tyr225. The specific activity of the EcOASS-B F141Y mutant toward OPS increased by 50-fold compared with that of the wild-type. Thus, a Tyr within a cysteine synthase corresponding to the Phe225 in ApOPSS and Phe141 in EcOASS-B could act as a key residue for classifying an unknown cysteine synthase as an OPSS. The elucidation of the substrate recognition system of cysteine synthases would enable us to effectively classify cysteine synthases and develop pathogen-specific drug targets, as OPSS is absent in mammalian hosts. [ABSTRACT FROM AUTHOR]

Abstract Background Acanthamoeba spp. can cause serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis and cutaneous acanthamoebiasis. Cysteine biosynthesis and the L-serine metabolic pathway play important roles in the energy metabolism of Acanthamoeba spp. However, no study has confirmed the functions of cysteine synthase (AcCS) in the cysteine pathway and phosphoglycerate dehydrogenase (AcGDH) or phosphoserine aminotransferase (AcSPAT) in the non-phosphorylation serine metabolic pathway of Acanthamoeba. Methods The AcCS, AcGDH and AcSPAT genes were amplified by PCR, and their recombinant proteins were expressed in Escherichia coli. Polyclonal antibodies against the recombinant proteins were prepared in mice and used to determine the subcellular localisation of each native protein by confocal laser scanning microscopy. The enzymatic activity of each recombinant protein was also analysed. Furthermore, each gene expression level was analysed by quantitative PCR after treatment with different concentrations of cysteine or L-serine. Results The AcCS gene encodes a 382-amino acid protein with a predicted molecular mass of 43.1 kDa and an isoelectric point (pI) of 8.11. The AcGDH gene encodes a 350-amino acid protein with a predicted molecular mass of 39.1 kDa and a pI of 5.51. The AcSPAT gene encodes a 354-amino acid protein with a predicted molecular mass of 38.3 kDa and a pI of 6.26. Recombinant AcCS exhibited a high cysteine synthesis activity using O-acetylserine and Na2S as substrates. Both GDH and SPAT catalysed degradation, rather than synthesis, of serine. Exogenous L-serine or cysteine inhibited the expression of all three enzymes in a time- and dose-dependent manner. Conclusions This study demonstrated that AcCS participates in cysteine biosynthesis and serine degradation via the non-phosphorylation serine metabolic pathway, providing a molecular basis for the discovery of novel anti-Acanthamoeba drugs.

Arsenic (As) contamination in paddy soil can cause phytotoxicity and elevated As accumulation in rice grains. Arsenic detoxification is closely linked to sulfur assimilation, but the genes involved have not been described in rice. In this study, we characterize the function of OASTL-A1, an O -acetylserine(thiol) lyase, in cysteine biosynthesis and detoxification of As in rice. Tissue expression analysis revealed that OsOASTL-A1 is mainly expressed in roots at the vegetative growth stage and in nodes at the reproductive stage. Furthermore, the expression of OsOASTL-A1 in roots was strongly induced by As exposure. Transgenic rice plants expressing pOsOASTL-A1::GUS (β-glucuronidase) indicated that OsOASTL-A1 was strongly expressed in the outer cortex and the vascular cylinder in the root mature zone. Subcellular localization using OsOASTL-A1:eGFP (enhanced green fluorescent protein) fusion protein showed that OsOASTL-A1 was localized to the cytosol. In vivo and in vitro enzyme activity assays showed that OsOASTL-A1 possessed the O -acetylserine(thiol) lyase activity. Knockout of OsOASTL-A1 led to significantly lower levels of cysteine, glutathione, and phytochelatins in roots and increased sensitivity to arsenate stress. Furthermore, the osoastl-a1 knockout mutants reduced As accumulation in the roots, but increased As accumulation in shoots. We conclude that OsOASTL-A1 is the cytosolic O -acetylserine(thiol) lyase that plays an important role in non-protein thiol biosynthesis in roots for As detoxification. [ABSTRACT FROM AUTHOR]

Cysteine is the main precursor of sulfur-containing biological molecules in bacteria and contributes to the control of the cell redox state. Hence, this amino acid plays an essential role in microbial survival and pathogenicity and the reductive sulfate assimilation pathway is considered a promising target for the development of new antibacterials. Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS-A), the enzymes catalyzing the last two steps of cysteine biosynthesis, engage in the formation of the cysteine synthase (CS) complex. The interaction between SAT and OASS-A finely tunes cysteine homeostasis, and the development of inhibitors targeting either protein–protein interaction or the single enzymes represents an attractive strategy to undermine bacterial viability. Given the peculiar mode of interaction between SAT and OASS-A, which exploits the insertion of SAT C-terminal sequence into OASS-A active site, we tested whether a recently developed competitive inhibitor of OASS-A exhibited any effect on the CS stability. Through surface plasmon resonance spectroscopy, we (i) determined the equilibrium constant for the Salmonella Typhimurium CS complex formation and (ii) demonstrated that the inhibitor targeting OASS-A active site affects CS complex formation. For comparison, the Escherichia coli CS complex was also investigated, with the aim of testing the potential broad-spectrum activity of the candidate antimicrobial compound.

Proteins utilizing pyridoxal 5'-phosphate as a coenzyme constitute a large superfamily and are currently classified into three functional groups and five structural fold types. Despite the variability of sequences and catalyzed reactions, they share relevant structural, dynamic and functional properties. Therefore, they constitute an optimal system to investigate the relative influence of primary sequence and coenzyme interactions on folding pathways, structural stability and enzymatic function. O-Acetylserine sulfhydrylase is a dimeric pyridoxal 5'-phosphate dependent enzyme that catalyzes the synthesis of L-cysteine from O-acetylserine and sulfide. The time-resolved fluorescence study of O-acetylserine sulfhydrylase unfolding, here reported, indicates that the coenzyme stabilizes the protein structure. The dependence on denaturant concentration of tryptophan lifetimes in the holo- and apo-enzyme demonstrates that the interactions with the coenzyme stabilize the C-terminal domain to a higher extent with respect to the N-terminal domain. This result is discussed in terms of a linkage between the differential stabilization brought about by the coenzyme and the different degrees of conformational flexibility required by the specialized functional role of distinct protein regions.

O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimurium, covalently binds one pyridoxal 5'-phosphate molecule per subunit as a fluorescent coenzyme. Different tautomers of the Schiff base between the coenzyme and lysine 41 generate structured absorption and fluorescence spectra upon one-photon excitation. We investigated the protein population heterogeneity by fluorescence correlation spectroscopy and lifetime techniques upon two-photon excitation. We sampled the fluorescence intensity from a small number of molecules (approximately 10) and analyzed the distribution of photon counts to separately determine the number and the fluorescence brightness of excited protein molecules. The changes in the average number of molecules and in the fluorescence brightness with the excitation wavelength indicate the presence of at least two fluorescent species, with two-photon excitation maxima at 660 and 800 nm. These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimine. Their relative abundance is estimated to be 4:1, whereas the ratio of their two-photon cross sections is reversed with respect to the single-photon excitation case. Consistent results are obtained from the measurement of the lifetime decays, which are sensitive to the excited-state heterogeneity. At least two components were detected, with lifetimes of approximately 2.5 and 0.5 ns. The lifetimes are very close to the values measured in bulk solutions upon one-photon excitation and attributed to the ketoenamine tautomer and to a dipolar species formed upon proton dissociation in the excited state.

Proteins belonging to the superfamily of pyridoxal 5'-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types. The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimensional structures of pyridoxal 5'-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the beta-family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo- and holoprotein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, (31)P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization. Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form. The data are discussed in light of the fact that the role of the coenzyme in structural stabilization varies among the pyridoxal 5'-phosphate-dependent enzymes and does not seem to be linked to the particular enzyme fold type.

Amebiasis is caused by infection with the protozoan parasite Entamoeba histolytica. Although metronidazole has been a drug of choice against amebiasis for decades, it shows side effects and low efficacy against asymptomatic cyst carriers. In addition, metronidazole resistance has been documented for bacteria and protozoa that share its targets, anaerobic energy metabolism. Therefore, drugs with new mode of action or targets are urgently needed. L-cysteine is the major thiol and an essential amino acid for proliferation and anti-oxidative defense of E. histolytica trophozoites. E. histolytica possesses the de novo L-cysteine biosynthetic pathway, consisting of two reactions catalyzed by serine acetyltransferase and cysteine synthase (CS, O-acetylserine sulfhydrylase). As the pathway is missing in humans, it is considered to be a rational drug target against amebiasis. In this study, we established a protocol to screen both a library of structurally known compounds and microbial culture extracts to discover compounds that target de novo cysteine biosynthesis of E. histolytica. The new screening system allowed us to identify the compounds that differentially affect the growth of the trophozoites in the cysteine-deprived medium compared to the cysteine-containing medium. A total of 431 structurally defined compounds of the Kitasato Natural Products Library and 6,900 microbial culture broth extracts were screened on the system described above. Five compounds, aspochalasin B, chaetoglobosin A, prochaetoglobosin III, cerulenin, and deoxyfrenolicin, from the Kitasato Natural Products Library, showed differential antiamebic activities in the cysteine-deprived medium when compared to the growth in the cysteine-containing medium. The selectivity of three cytochalasans apparently depends on their structural instability. Eleven microbial extracts showed selective antiamebic activities, and one fungal secondary metabolite, pencolide, was isolated. Pencolide showed cysteine deprivation-dependent antiamebic activity (7.6 times lower IC50 in the absence of cysteine than that in the presence of cysteine), although the IC50 value in the cysteine-deprived medium was rather high (283 μM). Pencolide also showed inhibitory activity against both CS1 and CS3 isoenzymes with comparable IC50 values (233 and 217 μM, respectively). These results indicated that antiamebic activity of pencolide is attributable to inhibition of CS. Cytotoxicity of pencolide was 6.7 times weaker against mammalian MRC-5 cell line than E. histotytica. Pencolide has the maleimide structure, which is easily attacked by Michael donors including the thiol moiety of cysteine. The cysteine-adducts of pencolide were detected by mass spectrometric analysis as predicted. As CS inhibition by the pencolide adducts was weak and their IC50 values to CS was comparable to that to the parasite in the cysteine-containing medium, the cysteine-adducts of pencolide likely contribute to toxicity of pencolide to the parasite in the cysteine-rich conditions. However, we cannot exclude a possibility that pencolide inactivates a variety of targets other than CSs in the absence of cysteine. Taken together, pencolide is the first compound that inhibits CS and amebic cell growth in a cysteine-dependent manner with relatively low mammalian cytotoxicity.

>bold<>italic/italic<>/bold< Systemic oxidative stress has been reported to play a central role in the pathogenesis of kidney function decline. The nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is one of the important endogenous antioxidant stress pathways in cells. This study aims to investigate whether shenduning granule can ameliorate oxidative stress in kidney tissues by activating the Nrf2/ARE pathway, and explores the detailed underlying mechanism. >bold<>italic/italic<>/bold< A total of 120 male Sprague-Dawley rats were randomly assigned to the sham-operated and operation groups. Rats in the operation group underwent 5/6 nephrectomy. Two weeks later, rats in the operation group were further randomly divided into 5 groups: model group, low-dose, medium-dose and high-dose shenduning granule groups, and losartan group. Rats in each group were given the same volume of corresponding liquid orally. Serum creatinine (SCr), blood urea nitrogen (BUN), 24-h urinary protein, malondialdehyde (MDA) and superoxide dismutase (SOD), Nrf2, heme oxygenase-1 (HO-1), and γ-glutamyl-cysteine synthetase (γ-GCS) were determined. >bold<>italic/italic<>/bold< Shenduning granule could markedly elevate HO-1, NRF2, γ-GCS and SOD (>italic/italic< < 0.05), and significantly decreased MDA, 24-h urinary protein, SCr and BUN in rats (>italic/italic< < 0.05). >bold<>italic/italic<>/bold< Shenduning granule can improve renal antioxidative stress activity in rats, exhibiting a renoprotective effect. The potential mechanism is likely exerted by the activation of the Nrf2/ARE pathway. [ABSTRACT FROM AUTHOR]

Cysteine persulfide and polysulfide are produced in cells and exist in abundance in both low MW and protein fractions. However, the mechanism of regulation of the formation of cellular cysteine polysulfides and the physiological functions of cysteine persulfides/polysulfides produced in cells are not fully understood. We recently demonstrated that cysteinyl-tRNA synthetase (CARS) is a novel cysteine persulfide synthase. CARS is involved in protein polysulfidation that is coupled with translation. In particular, mitochondria function in biogenesis and bioenergetics is also supported and up-regulated by cysteine persulfide derived from mitochondrial CARS (also known as CARS2). Here, we provide an overview of recent advances in reactive persulfide research and our understanding of the mechanisms underlying the formation and the physiological roles of reactive persufides, with a primary focus on the formation of cysteine persulfide by CARS and the most fundamental mitochondrial bioenergetics mediated by persulfides, that is, sulfur respiration. LINKED ARTICLES: This article is part of a themed section on Chemical Biology of Reactive Sulfur Species. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.4/issuetoc. [ABSTRACT FROM AUTHOR]

Abstract New mono-nuclear cobalt (II) tetra[4-(2-{(E)-[(4-bromophenyl)imino]methyl}phenoxy)] phthalocyanine (CoTBrImPc) complex has been synthesized for the first time in pure state. The synthesized dark blue coloured complex was characterized by elemental analysis, IR, UV–Vis, NMR, Mass, powder X-ray diffraction and thermogravimetric analysis (TGA) to understand the structural integrity, purity and crystalline property of the complex. The synthesized phthalocyanine complex was found to be electrochemically active and it was immobilized on the glassy carbon electrode by drop-casting method and used for the detection of l -cysteine. The CoTBrImPc modified electrode was found to be a good electrocatalyst for the catalytic oxidation of l -cysteine with shift in the overpotential towards less positive potential and an increase in the catalytic current compared to bare glassy carbon electrode (GCE). The linear response was observed for the voltametric detection of l -cysteine in the concentration range 10–100 nM with coefficient of regression R2 = 0.9993 and LOD of 3 nM and sensitivity of 2.99 μA nM−1 cm−2. The amperometric sensor was also developed for the detection of l -cysteine which showed linear response in the concentration range same as that of cyclic voltammertry technique with a linear equation y = 0.7582x + 16.9535 and correlation coefficient of R2 = 0.9961. The LOD and LOQ values for amperometric detection were 4 nM and 12 nM, respectively with the sensitivity value of 10.81 μA nM cm−2. Graphical abstract Unlabelled Image Highlights • New mono-nuclear CoTBrImPc complex has been synthesized in pure state. • The ligand and complex were characterized by spectroscopic and electrochemical techniques. • The CoTBrImPc modified electrode was employed for nanomolar detection of l -cysteine by voltammetry and amperometry. • The modified electrode is selective and sensitive for amperometric sensing of cysteine. • The modified electrode can be used for the analysis of cysteine in commercial sample. [ABSTRACT FROM AUTHOR]

Cysteine synthase A (CysK) catalyzes the last reaction of l‐cysteine synthesis in bacteria, but its moonlighting functions have been revealed recently. In this study, CysK was overexpressed in Corynebacterium glutamicum IWJ001, an l‐isoleucine producer. Compared with the control IWJ001/pDXW‐8, IWJ001/pDXW‐8‐cysK cells grew fast during log phase, and produced 26.5% more l‐isoleucine in flask fermentation and 23.5% more l‐isoleucine in fed‐batch fermentation. The key genes aspC, lysC, hom, thrB, ilvA, and ilvBN involved in l‐isoleucine biosynthesis were all upregulated in IWJ001/pDXW‐8‐cysK, compared with IWJ001/pDXW‐8. In addition, IWJ001/pDXW‐8‐cysK cells were longer and thicker than IWJ001/pDXW‐8 cells. Compared with IWJ001/pDXW‐8, the membrane permeability increased 15.8% and biofilm formation ability decreased 71.3% for IWJ001/pDXW‐8‐cysK cells. The results demonstrate that CysK overexpression in C. glutamicum is a good approach to enhance l‐isoleucine production. [ABSTRACT FROM AUTHOR]

Several bacteria rely on the reductive sulphur assimilation pathway, absent in mammals, to synthesise cysteine. Reduction of virulence and decrease in antibiotic resistance have already been associated with mutations on the genes that codify cysteine biosynthetic enzymes. Therefore, inhibition of cysteine biosynthesis has emerged as a promising strategy to find new potential agents for the treatment of bacterial infection. Following our previous efforts to explore OASS inhibition and to expand and diversify our library, a scaffold hopping approach was carried out, with the aim of identifying a novel fragment for further development. This novel chemical tool, endowed with favourable pharmacological characteristics, was successfully developed, and a preliminary Structure-Activity Relationship investigation was carried out. [ABSTRACT FROM AUTHOR]

O-acetylserine sulfhydrylase (OASS) is the pyridoxal 5′-phosphate dependent enzyme that catalyses the formation of L-cysteine in bacteria and plants. Its inactivation is pursued as a strategy for the identification of novel antibiotics that, targeting dispensable proteins, holds a great promise for circumventing resistance development. In the present study, we have investigated the reactivity of Salmonella enterica serovar Typhimurium OASS-A and OASS-B isozymes with fluoroalanine derivatives. Monofluoroalanine reacts with OASS-A and OASS-B forming either a stable or a metastable α-aminoacrylate Schiff’s base, respectively, as proved by spectral changes. This finding indicates that monofluoroalanine is a substrate analogue, as previously found for other beta-halogenalanine derivatives. Trifluoroalanine caused different and time-dependent absorbance and fluorescence spectral changes for the two isozymes and is associated with irreversible inhibition. The time course of enzyme inactivation was found to be characterised by a biphasic behaviour. Partially distinct inactivation mechanisms for OASS-A and OASS-B are proposed. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae is known to grow with thiosulfate as a sulfur source, and it produces more ethanol when using thiosulfate than using sulfate. Here, we report how it assimilates thiosulfate. S. cerevisiae absorbed thiosulfate into the cell through two sulfate permeases, Sul1 and Sul2. Two rhodaneses, Rdl1 and Rdl2, converted thiosulfate to a persulfide and sulfite. The persulfide was reduced by cellular thiols to H2S, and sulfite was reduced by sulfite reductase to H2S. Cysteine synthase incorporated H2S into O-acetyl-L-homoserine to produce L-homocysteine, which is the precursor for cysteine and methionine in S. cerevisiae. Several other rhodaneses replaced Rdl1 and Rdl2 for thiosulfate utilization in the yeast. Thus, any organisms with the sulfate assimilation system potentially could use thiosulfate as a sulfur source, since rhodaneses are common in most organisms. IMPORTANCE The complete pathway of thiosulfate assimilation in baker's yeast is determined. The finding reveals the extensive overlap between sulfate and thiosulfate assimilation. Rhodanese is the only additional enzyme for thiosulfate utilization. The common presence of rhodanese in most organisms, including Bacteria, Archaea, and Eukarya, suggests that most organisms with the sulfate assimilation system also use thiosulfate. Since it takes less energy to reduce thiosulfate than sulfate for assimilation, thiosulfate has the potential to become a choice of sulfur in optimized media for industrial fermentation. [ABSTRACT FROM AUTHOR]

Amino acids (AAs) play significant roles in metal binding, antioxidant defense, and signaling in plants during heavy metal stress. In the present study, the essential amino acids (EAAs), non-essential amino acids (NEAAs), as well as the enzymes of proline and cysteine biosynthetic pathways were studied in contrasting arsenic accumulating rice genotypes grown in hydroponic solutions with addition of arsenate (AsV) or arsenite (AsIII). Under a mild As stress, the total AAs content significantly increased in both the rice genotypes with a greater increase in a low As accumulating rice genotype (LAARG; IET-19226) than in a high As accumulating rice genotype (HAARG; BRG-12). At the equimolar concentration (10 μM), AsIII had a greater effect on EAAs than AsV. Conversely, AsV was more effective in inducing a proline accumulation than AsIII. Among NEAAs, As significantly induced the accumulation of histidine, aspartic acid, and serine. In contrast, a higher As concentration (50 μM) reduced the content of most AAs, the effect being more prominent during AsIII exposure. The inhibition of glutamate kinase activity was noticed in HAARG, conversely, serine acetyltransferase and cysteine synthase activities were increased which was positively correlated with the cysteine synthesis.

Garlic is a valuable source of healthy compounds, including secondary metabolites rich in sulphur such as cysteine sulphoxides (CSOs). Here, we present new qRT-PCR assays analysing the transcription of two genes encoding key enzymes in CSO biosynthetic pathways (cysteine synthase and alliinase) in developing garlic. We also identified a set of genes (ACT I, GAPDH, and TUB) to use as transcription normalisation controls. We showed that the (normalised) transcription of both enzymes was highest during sprouting and decreased significantly in fully developed leaves, which are the major CSO-producing organs. Transcriptional activity further declined at the end of the growing season. Different cultivars show similar sulphur metabolism gene expression when European garlics were compared to Chinese and American genotypes. The qRT-PCR assays presented are also suitable for investigating the effects of agricultural practices on CSO formation in garlic to satisfy consumer demands. [ABSTRACT FROM AUTHOR]

We elucidated a unique feature of sulfur metabolism in Cryptococcus neoformans. C. neoformans produces cysteine solely by the O-acetylserine pathway that consists of serine-O-acetyl transferase and cysteine synthase. We designated the gene encoding the former enzyme CYS2 (locus tag CNE02740) and the latter enzyme CYS1 (locus tag CNL05880). The cys1Δmutant strain was found to be avirulent in a murine infection model. Methionine practically does not support growth of the cys1Δ strain, and cysteine does not serve as a methionine source, indicating that the transsulfuration pathway does not contribute to sulfur amino acid synthesis in C. neoformans. Among the genes encoding enzymes catalyzing the reactions from homoserine to methionine, the gene corresponding to the Saccharomyces cerevisiae MET17 encoding O-acetylhomoserine sulfhydrylase (Met17p) had remained to be identified in C. neoformans. By genetic analysis of Met− mutants obtained by Agrobacterium tumefaciens-mediated mutagenesis, we concluded that Cnc01220, most similar to Str2p (36% identity), cystathionine-γ-synthase, in the Saccharomyces genome, is the C. neoformans version of O-acetylhomoserine sulfhydrylase. We designated CNC01220 as MET17. The C. neoformans met3Δ mutant defective in the first step of the sulfate assimilation pathway, sulfate adenylyltransferase, barely uses methionine as a sulfur source, whereas it uses cysteine efficiently. The poor utilization of methionine by the met3Δ mutant is most probably due to the absence of the transsulfuration pathway, causing an incapability of C. neoformans to produce cysteine and hydrogen sulfide from methionine. When cysteine is used as a sulfur source, methionine is likely produced de novo by using hydrogen sulfide derived from cysteine via an unidentified pathway. Altogether, the unique features of sulfur amino acid metabolism in C. neoformans will make this fungus a valuable experimental system to develop anti-fungal agents and to investigate physiology of hydrogen sulfide. [ABSTRACT FROM AUTHOR]

Drug resistance to almost all antibiotics of Shigella flexneri , a major cause of shigellosis in developing countries, necessitates continuous discovery of novel therapeutics. This study reports a structure-function analysis of a potential drug target serine acetyltransferase (CysE), an enzyme of de novo cysteine biosynthesis pathway that is absent in humans. Analysis of CysE sequences of S. flexneri species and serotypes displayed only two variants that differed by a single amino acid substitution at position 241. Structural inspection of the available crystal structure disclosed this site to be distinct from the substrate/cofactor binding pockets or dimer/trimer interfaces. This study discovers that V241 variant of S. flexneri CysE has nearly null enzymatic activity. The observation is explained by molecular dynamic studies which reveal that the disorder generated by A241V substitution is the basis of dissociation of the quaternary assembly of S. flexneri CysE leading to loss of enzymatic activity and stability. The study provides the first evidence that position 241 of CysE, affects the catalytic efficiency of enzyme and suggests this locus as a ‘hot spot’ for the propagation of conformational changes. It may be postulated that transient quaternary structure of CysE maybe another mechanism for regulating the intracellular level of cysteine. [ABSTRACT FROM AUTHOR]

Aims This study uncovered that the genetically endowed intracellular glutathione contents (iGSH) regulated by the catalytic subunit of γ‑glutamylcysteine synthetase heavy chain (γ‑GCSh) as a prime target for overcoming both the inherited and stimuli-activated chemo- and radio-resistance of hepatocellular carcinoma (HCC) cells. Main methods Reactive oxygen species (ROS) production and mitochondrial membrane potential (Δψm) were determined by the probe-based flow cytometry. The TUNEL assay was used as an index of radio-sensitivity and the MTT assay was used as an index of chemo-sensitivity against various anti-cancer agents. iGSH and γ‑GCSh activity were measured by HPLC methods. γ‑GCSh-overexpressing GCS30 cell line was established by tetracycline-controlled Tet-OFF gene expression system in SK-Hep-1 cells. Key findings The relative radio-sensitivities of a panel of five HCC cells were found to be correlated negatively with both the contents of iGSH and their corresponding γ‑GCSh activities with an order of abundance being Hep G2 > Hep 3B > J5 > Mahlavu > SK-Hep-1, respectively. Similarly, the cytotoxicity response patterns of these HCC cells against arsenic trioxide (ATO), a ROS-producing anti-cancer drug, were exactly identical to the order of ranking instigated by the radiotherapy (RT) treatment. Next, γ‑GCSh-overexpressing GCS30 cells were found to possess excellent ability to profoundly mitigate both the drop of Δψm and apoptotic TUNEL-positive cell population engendered by ATO, cisplatin, doxorubicin, and RT treatments. Significance Our data unequivocally demonstrate that γ‑GCSh may represent a prime target for overcoming anti-cancer drugs and RT resistance for HCC cells. [ABSTRACT FROM AUTHOR]

In the cysteine and mimosine biosynthesis process, O-acetyl-l-serine (OAS) is the common substrate. In the presence of O-acetylserine (thiol) lyase (OASTL, cysteine synthase) the reaction of OAS with sulfide produces cysteine, while with 3-hydroxy-4-pyridone (3H4P) produces mimosine. The enzyme OASTL can either catalyze Cys synthesis or both Cys and mimosine. A cDNA for cytosolic OASTL was cloned from M. pudica for the first time containing 1,410 bp nucleotides. The purified protein product from overexpressed bacterial cells produced Cys only, but not mimosine, indicating it is Cys specific. Kinetic studies revealed that pH and temperature optima for Cys production were 6.5 and 50 °C, respectively. The measured Km, Kcat, and Kcat Km−1 values were 159 ± 21 µM, 33.56 s−1, and 211.07 mM−1s−1 for OAS and 252 ± 25 µM, 32.99 s−1, and 130.91 mM−1s−1 for Na2S according to the in vitro Cys assay. The Cy-OASTL of Mimosa pudica is specific to Cys production, although it contains sensory roles in sulfur assimilation and the reduction network in the intracellular environment of M. pudica. [ABSTRACT FROM AUTHOR]

Main conclusion Different levels of salt stress affected the OAS-TL expression levels in Pancratium maritimum organs (bulb, leaf and root). A detailed method has been described for the identification of the conserved domain of the OASTL cDNA in sea daffodil given the scarce data available for the Amaryllidaceae family. Pancratium maritimum or sea daffodil (Amaryllidaceae) is a bulbous geophyte growing on coastal sands. In this study, we investigated the involvement of cysteine synthesis for salt tolerance through the expression of the enzyme O-acetylserine(thio) lyase (OAS-TL) during the stress response to NaCl treatments in P. maritimum. Quantitative real-time PCR was used in different organs (bulb, leaf and root). [ABSTRACT FROM AUTHOR]

Grass pea ( Lathyrus sativus L.) is a worldwide popular pulse crop especially for its protein rich seeds with least production cost. However, the use of the crop became controversial due to the presence of non-protein amino acid, β-N-oxalyl-L-α, β-diaminopropionic acid (β-ODAP) in its seed and leaf, which is known as the principle neurotoxin to cause neurolathyrism (a motor neurodegenerative disease of humans and animals) during prolonged consumption as regular diet. Till date, the knowledge on β-ODAP biosynthesis in Lathyrus sp. is limited only to a small part of the complex bio-chemical steps involved including a few known sulfur-containing enzymes (viz. cysteine synthase , ODAP synthase etc.). In Lathyrus sativus , biosynthesis of β-ODAP varies differentially in a tissue-specific manner as well as in response to several environmental stresses viz. zinc deficiency, iron over-exposure, moisture stress etc. In the present study, a novel cysteine synthase gene ( LsCSase ) from Lathyrus sativus L was identified and characterized through bioinformatics approaches. The bioinformatic analysis revealed that LsCSase showed maximum similarity with the O-acetyl serine (thiol) lyase of Medicago truncatula with respect to several significant sequence-specific conserved motifs (cysK, CBS like, ADH_zinc_N, PALP), sub-cellular localization (chloroplast or cytoplasm) etc., similar to other members of cysteine synthase protein family. Moreover, the tissue-specific regulation of the LsCSase as well as its transcriptional activation under certain previously reported stressed conditions (low Zn +2 -high Fe +2 , PEG induced osmotic stress) were also documented through quantitative real-time PCR analyses, suggesting a possible link between the LsCSase gene activation and β-ODAP biosynthesis to manage external stresses in grass pea. This preliminary study offers a probable way towards the development of less toxic consumer-safe grass pea by down-regulation or deactivation of such gene/s ( cysteine synthase ) through genetic manipulations. [ABSTRACT FROM AUTHOR]

Mycothiol cysteine ligase (MshC) is a key enzyme in the mycothiol (MSH) biosynthesis and a promising target for developing new anti-mycobacterial compounds. Herein, we report on the synthesis of substrate analogues, as potential inhibitors, for the MshC enzyme. The target molecules were synthesized employing a Schmidt glycosylation strategy using an enantiomerically pure inositol acceptor and 2-deoxy trichloroacetimidate glycosyl donors with glycosylation yields greater than 70% and overall yields >5%. The inositol acceptor was obtained via chiral resolution of (±)- myo -inositol. [ABSTRACT FROM AUTHOR]

Abstract Background Cysteine, a sulfur-containing amino acid, plays an important role in a variety of cellular functions such as protein biosynthesis, methylation, and polyamine and glutathione syntheses. In trypanosomatids, glutathione is conjugated with spermidine to form the specific antioxidant thiol trypanothione (T[SH]2) that plays a central role in maintaining intracellular redox homeostasis and providing defence against oxidative stress. Methods We cloned and characterised genes coding for a cystathionine β-synthase (CβS) and cysteine synthase (CS), key enzymes of the transsulfuration and assimilatory pathways, respectively, from the hemoflagellate protozoan parasite Trypanosoma rangeli. Results Our results show that T. rangeli CβS (TrCβS), similar to its homologs in T. cruzi, contains the catalytic domain essential for enzymatic activity. Unlike the enzymes in bacteria, plants, and other parasites, T. rangeli CS lacks two of the four lysine residues (Lys26 and Lys184) required for activity. Enzymatic studies using T. rangeli extracts confirmed the absence of CS activity but confirmed the expression of an active CβS. Moreover, CβS biochemical assays revealed that the T. rangeli CβS enzyme also has serine sulfhydrylase activity. Conclusion These findings demonstrate that the RTS pathway is active in T. rangeli, suggesting that this may be the only pathway for cysteine biosynthesis in this parasite. In this sense, the RTS pathway appears to have an important functional role during the insect stage of the life cycle of this protozoan parasite.