Mutational analysis of Sep-tRNA:Cys-tRNA synthase reveals critical residues for tRNA-dependent cysteine formation

In methanogenic archaea, Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). The mechanism of tRNA-dependent cysteine formation remains unclear due to the lack of functional studies. In this work, we mutated 19 conserved residues in Methanocaldococcus jannaschii SepCysS, and employed an in vivo system to determine the activity of the resulting variants. Our results show that three active-site cysteines (Cys39, Cys42 and Cys247) are essential for SepCysS activity. In addition, combined with structural modeling, our mutational and functional analyses also reveal multiple residues that are important for the binding of PLP, Sep and tRNA. Our work thus represents the first systematic functional analysis of conserved residues in archaeal SepCysSs, providing insights into the catalytic and substrate binding mechanisms of this poorly characterized enzyme.     

Editing function of Escherichia coli cysteinyl-tRNA synthetase: cyclization of cysteine to cysteine thiolactone.

A cyclic sulfur compound, identified as cysteine thiolactone by several chemical and enzymatic tests, is formed from cysteine during in vitro tRNA(Cys) aminoacylation catalyzed by Escherichia coli cysteinyl-tRNA synthetase. The mechanism of cysteine thiolactone formation involves enzymatic deacylation of Cys-tRNA(Cys) (k = 0.017 s-1) in which nucleophilic sulfur of the side chain of cysteine in Cys-tRNA(Cys) attacks its carboxyl carbon to yield cysteine thiolactone. Nonenzymatic deacylation of Cys-tRNA(Cys) (k = 0.0006 s-1) yields cysteine, as expected. Inhibition of enzymatic deacylation of Cys-tRNA(Cys) by cysteine and Cys-AMP, but not by ATP, indicates that both synthesis of Cys-tRNA(Cys) and cyclization of cysteine to the thiolactone occur in a single active site of the enzyme. The cyclization of cysteine is mechanistically similar to the editing reactions of methionyl-tRNA synthetase. However, in contrast to methionyl-tRNA synthetase which needs the editing function to reject misactivated homocysteine, cysteinyl-tRNA synthetase is highly selective and is not faced with a problem in rejecting noncognate amino acids. Despite this, the present day cysteinyl-tRNA synthetase, like methionyl-tRNA synthetase, still retains an editing activity toward the cognate product, the charged tRNA. This function may be a remnant of a chemistry used by an ancestral cysteinyl-tRNA synthetase.     

Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe-S cluster and thionucleoside biosynthesis in vivo

IscS catalyzes the fragmentation of l-cysteine to l-alanine and sulfane sulfur in the form of a cysteine persulfide in the active site of the enzyme. In Escherichia coli IscS, the active site cysteine Cys(328) resides in a flexible loop that potentially influences both the formation and stability of the cysteine persulfide as well as the specificity of sulfur transfer to protein substrates. Alanine-scanning substitution of this 14 amino acid region surrounding Cys(328) identified additional residues important for IscS function in vivo. Two mutations, S326A and L333A, resulted in strains that were severely impaired in Fe-S cluster synthesis in vivo. The mutant strains were deficient in Fe-S cluster-dependent tRNA thionucleosides (s(2)C and ms(2)i(6)A) yet showed wild type levels of Fe-S-independent thionucleosides (s(4)U and mnm(5)s(2)U) that require persulfide formation and transfer. In vitro, the mutant proteins were similar to wild type in both cysteine desulfurase activity and sulfur transfer to IscU. These results indicate that residues in the active site loop can selectively affect Fe-S cluster biosynthesis in vivo without detectably affecting persulfide delivery and suggest that additional assays may be necessary to fully represent the functions of IscS in Fe-S cluster formation.     

Structural insights into the second step of RNA-dependent cysteine biosynthesis in archaea: crystal structure of Sep-tRNA:Cys-tRNA synthase from Archaeoglobus fulgidus

In the ancient organisms, methanogenic archaea, lacking the canonical cysteinyl-tRNA synthetase, Cys-tRNA(Cys) is produced by an indirect pathway, in which O-phosphoseryl-tRNA synthetase ligates O-phosphoserine (Sep) to tRNA(Cys) and Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). In this study, the crystal structure of SepCysS from Archaeoglobus fulgidus has been determined at 2.4 A resolution. SepCysS forms a dimer, composed of monomers bearing large and small domains. The large domain harbors the seven-stranded beta-sheet, which is typical of the pyridoxal 5'-phosphate (PLP)-dependent enzymes. In the active site, which is located near the dimer interface, PLP is covalently bound to the side-chain of the conserved Lys209. In the proximity of PLP, a sulfate ion is bound by the side-chains of the conserved Arg79, His103, and Tyr104 residues. The active site is located deep within the large, basic cleft to accommodate Sep-tRNA(Cys). On the basis of the surface electrostatic potential, the amino acid residue conservation mapping, the position of the bound sulfate ion, and the substrate amino acid binding manner in other PLP-dependent enzymes, a binding model of Sep-tRNA(Cys) to SepCysS was constructed. One of the three strictly conserved Cys residues (Cys39, Cys42, or Cys247), of one subunit may play a crucial role in the catalysis in the active site of the other subunit.     

Redundant synthesis of cysteinyl-tRNACys in Methanosarcina mazei

A subset of methanogenic archaea synthesize the cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) needed for protein synthesis using both a canonical cysteinyl-tRNA synthetase (CysRS) as well as a set of two enzymes that operate via a separate indirect pathway. In the indirect route, phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)) is first synthesized by phosphoseryl-tRNA synthetase (SepRS), and this misacylated intermediate is then converted to Cys-tRNA(Cys) by Sep-tRNA:Cys-tRNA synthase (SepCysS) via a pyridoxal phosphate-dependent mechanism. Here, we explore the function of all three enzymes in the mesophilic methanogen Methanosarcina mazei. The genome of M. mazei also features three distinct tRNA(Cys) isoacceptors, further indicating the unusual and complex nature of Cys-tRNA(Cys) synthesis in this organism. Comparative aminoacylation kinetics by M. mazei CysRS and SepRS reveals that each enzyme prefers a distinct tRNA(Cys) isoacceptor or pair of isoacceptors. Recognition determinants distinguishing the tRNAs are shown to reside in the globular core of the molecule. Both enzymes also require the S-adenosylmethione-dependent formation of (m1)G37 in the anticodon loop for efficient aminoacylation. We further report a new, highly sensitive assay to measure the activity of SepCysS under anaerobic conditions. With this approach, we demonstrate that SepCysS functions as a multiple-turnover catalyst with kinetic behavior similar to bacterial selenocysteine synthase and the archaeal/eukaryotic SepSecS enzyme. Together, these data suggest that both metabolic routes and all three tRNA(Cys) species in M. mazei play important roles in the cellular physiology of the organism.     

Methanocaldococcus jannaschii prolyl-tRNA synthetase charges tRNA(Pro) with cysteine

Methanocaldococcus jannaschii prolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) to make up for the absence of the canonical cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H., Ibba, M., and S?ll, D. (2000) Science 287, 479-482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000) Biochemistry 39, 7792-7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinant M. jannaschii ProRS misaminoacylates M. jannaschii tRNA(Pro) with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschii tRNA(Cys) with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludis cysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNA(Pro) readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNA(Cys) in M. jannaschii.     

The glial Gomori-positive material is sulfane sulpfur

The Gomori-positive glia are periventricular astrocytes with abundant cytoplasmic granular material, predominantly occupying a periventricular site in the brain. These granular inclusions are strongly stained with chrome hematoxylin in the Gomori's method as well as exhibit red autofluorescence and non-enzymatic peroxidase activity. The glial Gomori-positive material (GGPM) granules are positive in the performic acid Alcian blue method indicating the presence of protein-bound sulfur, what has been shown by our previous studies. The number of cells containing glial Gomori-positive granules dropped after administration of cyanide and increased under the influence of sulfane sulfur donor (diallyl disulfide). This suggests, that sulfur of these granules is a sulfane sulfur, possibly in the form of protein-bound cysteine persulfide. Sulfane sulfur is labile, reactive sulfur atom covalently bound to another sulfur atom. In this paper we present evidence that GGPM exhibit affinity to cyanolysis and its stainability in Gomori's method is due to the presence of protein-bound sulfane sulfur. The biological role of the Gomori-positive glia connected with protective properties of sulfane sulfur has been discussed.     

Persulfide generated from L-cysteine inactivates tyrosine aminotransferase. Requirement for a protein with cysteine oxidase activity and gamma-cystathionase

Liver cytosols contain factors that produce an inhibitor of tyrosine aminotransferase and other enzymes when incubated with L-cysteine or L-cystine. Cystine-dependent inactivation was caused by cystathionase and required pyridoxal 5'-phosphate, but a second protein was needed to reconstitute cysteine-dependent inactivation. A cytosolic protein was isolated that oxidized free cysteine and brought about inactivation of tyrosine aminotransferase when coincubated with cystathionase. Hematin also oxidized cysteine, which led to cysteine-dependent inactivation of tyrosine aminotransferase in the presence of cystathionase. The inactivation of tyrosine aminotransferase involved three steps: initial oxidation of cysteine to form cystine; desulfuration of cystine catalyzed by cystathionase to form the persulfide, thiocysteine; and reaction of thiocysteine (or products of its decomposition) with proteins to form protein-bound sulfane. Since dithiothreitol reactivated tyrosine aminotransferase, the sulfane probably inactivated the enzyme by oxidation of thiol groups. The present results do not indicate whether the cysteine oxidase activity is enzymatic nor do they prove which form of polysulfide inactivates tyrosine aminotransferase. Reduced glutathione greatly slowed the rates at which sulfane accumulated and at which tyrosine aminotransferase was inactivated. Incubation of DL-cystathionine with liver cytosols led to formation of cysteine, which was oxidized and cleaved to form persulfide, and caused inactivation of tyrosine aminotransferase. Thus, sulfane sulfur that is generated by an enzyme of the transulfuration pathway inactivates a transaminase by nonselective oxidation of enzyme-bound thiol groups.     

Assembly of iron-sulfur clusters mediated by cysteine desulfurases,IscS, CsdB and CSD,from Escherichia coli

Cysteine desulfurase plays a principal role in the assembly of iron-sulfur clusters by mobilizing the sulfur atom of L-cysteine. The active site cysteine residue of the enzyme attacks the sulfur atom of L-cysteine to form a cysteine persulfide residue, and the substrate-derived sulfur atom of this residue is incorporated into iron-sulfur clusters. Escherichia coli has three cysteine desulfurases named IscS, CsdB and CSD. We found that each of them facilitates the formation of the iron-sulfur cluster of ferredoxin in vitro. Since IscU, an iron-sulfur protein of E. coli, is believed to function as a scaffold for the cluster assembly in vivo, we examined whether IscS, CsdB and CSD interact with IscU to deliver the sulfur atom to IscU. By surface plasmon resonance analysis, we found that only IscS interacts with IscU. We isolated the IscS/IscU complex, determined the residues involved in the formation of the complex, and obtained data suggesting that the sulfur transfer from IscS to IscU is initiated by the attack of Cys63 of IscU on the S gamma atom of the cysteine persulfide residue transiently produced on IscS.     

Primary structure of human copper-zinc superoxide dismutase. Cysteine - and tryptophan - containing peptides

Analysis of soluble peptides derived from tryptic and chymotryptic digestions of carboxymethylated human superoxide dismutase gives primary structural information for approximately 67% of the protein. Regions so far elucidated appear to be highly homologous to the corresponding bovine enzyme; in particular Cys 6 and the two cysteine residues 55 and 144, which form the intrasubunit disulfide bond of the bovine enzyme, are conserved. A cluster of three substitutions including the fourth cysteine residue unique to the human enzyme has been found in positions 107–109 and may be related to the presence of persulfide groups in the human enzyme. The single tryptophan residue of the human protein is not homologous to the single tyrosine residue of the bovine protein.     

Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis

The Moco (molybdenum cofactor) sulfurase ABA3 from Arabidopsis thaliana catalyses the sulfuration of the Moco of aldehyde oxidase and xanthine oxidoreductase, which represents the final activation step of these enzymes. ABA3 consists of an N-terminal NifS-like domain that exhibits L-cysteine desulfurase activity and a C-terminal domain that binds sulfurated Moco. The strictly conserved Cys430 in the NifS-like domain binds a persulfide intermediate, which is abstracted from the substrate L-cysteine and finally needs to be transferred to the Moco of aldehyde oxidase and xanthine oxidoreductase. In addition to Cys?3?, another eight cysteine residues are located in the NifS-like domain, with two of them being highly conserved among Moco sulfurase proteins and, at the same time, being in close proximity to Cys?3?. By determination of the number of surface-exposed cysteine residues and the number of persulfide-binding cysteine residues in combination with the sequential substitution of each of the nine cysteine residues, a second persulfide-binding cysteine residue, Cys2??, was identified. Furthermore, the active-site Cys?3? was found to be located on top of a loop structure, formed by the two flanking residues Cys?2? and Cys?3?, which are likely to form an intramolecular disulfide bridge. These findings are confirmed by a structural model of the NifS-like domain, which indicates that Cys?2? and Cys?3? are within disulfide bond distance and that a persulfide transfer from Cys?3? to Cys2?? is indeed possible.     

Evidence for the transfer of sulfane sulfur from IscS to ThiI during the in vitro biosynthesis of 4-thiouridine in Escherichia coli tRNA

IscS from Escherichia coli is a cysteine desulfurase that has been shown to be involved in Fe-S cluster formation. The enzyme converts L-cysteine to L-alanine and sulfane sulfur (S(0)) in the form of a cysteine persulfide in its active site. Recently, we reported that IscS can donate sulfur for the in vitro biosynthesis of 4-thiouridine (s(4)U), a modified nucleotide in tRNA. In addition to IscS, s(4)U synthesis in E. coli also requires the thiamin biosynthetic enzyme ThiI, Mg-ATP, and L-cysteine as the sulfur donor. We now report evidence that the sulfane sulfur generated by IscS is transferred sequentially to ThiI and then to tRNA during the in vitro synthesis of s(4)U. Treatment of ThiI with 5-((2-iodoacetamido)ethyl)-1-aminonapthalene sulfonic acid (I-AEDANS) results in irreversible inhibition, suggesting the presence of a reactive cysteine that is required for binding and/or catalysis. Both ATP and tRNA can protect ThiI from I-AEDANS inhibition. Finally, using gel shift and protease protection assays, we show that ThiI binds to unmodified E. coli tRNA(Phe). Together, these results suggest that ThiI is a recipient of S(0) from IscS and catalyzes the ultimate sulfur transfer step in the biosynthesis of s(4)U.     

Persulfide properties of thiocystine and related trisulfides

Thiocystine (bis[2-amino-2-carboxyethyl]trisulfide) functions as a persulfide in transferring its sulfane sulfur to thiophilic acceptors. This occurs by formation of a reactive intermediate, thiocysteine (alanine hydrogen disulfide). In the absence of an acceptor sulfur is released in elemental form. Thiocystine is relatively stable in the pH range of 2–9. However, its conversion to unstable thiocysteine is accelerated by sulfhydryl compounds, rhodanese, or reagents that cleave sulfur-sulfur bonds to yield sulfhydryl groups. Since thiocystine has been detected in biological systems, it is proposed that in provides a storage form of sulfane sulfur. Trisulfides related to thiocystine show qualitatively similar properties.     

Bacterial cysteine desulfurases: their function and mechanisms

Cysteine desulfurase is a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme that catalyzes the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. Increased evidence for the functions of cysteine desulfurases has revealed their important roles in the biosyntheses of Fe-S clusters, thiamine, thionucleosides in tRNA, biotin, lipoic acid, molybdopterin, and NAD. The enzymes are also proposed to be involved in cellular iron homeostasis and in the biosynthesis of selenoproteins. The mechanisms for sulfur mobilization mediated by cysteine desulfurases are as yet unknown, but enzymes capable of providing a variety of biosynthetic pathways for sulfur/selenium-containing biomolecules are probably applicable to the production of cofactors and the bioconversion of useful compounds.     

Role of conserved cysteines in mediating sulfur transfer from IscS to IscU

The role of the three conserved cysteine residues on Azotobacter vinelandii IscU in accepting sulfane sulfur and forming a covalent complex with IscS has been evaluated using electrospray-ionization mass spectrometry studies of variants involving individual cysteine-to-alanine substitutions. The results reveal that IscS can transfer sulfur to each of the three alanine-substituted forms of IscU to yield persulfide or polysulfide species, and formation of a heterodisulfide covalent complex between IscS and Cys(37) on IscU. It is concluded that S transfer from IscS to IscU does not involve a specific cysteine on IscU or the formation of an IscS-IscU heterodisulfide complex.     

Cysteinyl-tRNA(Cys) formation in Methanocaldococcus jannaschii: the mechanism is still unknown

Most organisms form Cys-tRNA(Cys), an essential component for protein synthesis, through the action of cysteinyl-tRNA synthetase (CysRS). However, the genomes of Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, and Methanopyrus kandleri do not contain a recognizable cysS gene encoding CysRS. It was reported that M. jannaschii prolyl-tRNA synthetase (C. Stathopoulos, T. Li, R. Longman, U. C. Vothknecht, H. D. Becker, M. Ibba, and D. S?ll, Science 287:479-482, 2000; R. S. Lipman, K. R. Sowers, and Y. M. Hou, Biochemistry 39:7792-7798, 2000) or the M. jannaschii MJ1477 protein (C. Fabrega, M. A. Farrow, B. Mukhopadhyay, V. de Crécy-Lagard, A. R. Ortiz, and P. Schimmel, Nature 411:110-114, 2001) provides the "missing" CysRS activity for in vivo Cys-tRNA(Cys) formation. These conclusions were supported by complementation of temperature-sensitive Escherichia coli cysS(Ts) strain UQ818 with archaeal proS genes (encoding prolyl-tRNA synthetase) or with the Deinococcus radiodurans DR0705 gene, the ortholog of the MJ1477 gene. Here we show that E. coli UQ818 harbors a mutation (V27E) in CysRS; the largest differences compared to the wild-type enzyme are a fourfold increase in the K(m) for cysteine and a ninefold reduction in the k(cat) for ATP. While transformants of E. coli UQ818 with archaeal and bacterial cysS genes grew at a nonpermissive temperature, growth was also supported by elevated intracellular cysteine levels, e.g., by transformation with an E. coli cysE allele (encoding serine acetyltransferase) or by the addition of cysteine to the culture medium. An E. coli cysS deletion strain permitted a stringent complementation test; growth could be supported only by archaeal or bacterial cysS genes and not by archaeal proS genes or the D. radiodurans DR0705 gene. Construction of a D. radiodurans DR0705 deletion strain showed this gene to be dispensable. However, attempts to delete D. radiodurans cysS failed, suggesting that this is an essential Deinococcus gene. These results imply that it is not established that proS or MJ1477 gene products catalyze Cys-tRNA(Cys) synthesis in M. jannaschii. Thus, the mechanism of Cys-tRNA(Cys) formation in M. jannaschii still remains to be discovered.     

Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase

The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active. Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition. In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents. In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated beta-(4-hydroxyphenyl)ethyl maleimide. These are located in the large domain of the homodimer, about 10 A from the active site. The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318. Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 A), requiring only a decrease of 1.1-1.5 A. In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm. Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5'-dithiobis(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein. Furthermore, it was established that reaction of hBCATm with H2O2 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318. Addition of dithiothreitol completely reversed the oxidation and restored activity. Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center. Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism.     

Bacterial cysteine desulfurases: versatile key players in biosynthetic pathways of sulfur-containing biofactors

Cysteine desulfurases are pyridoxal 5′-phosphate-dependent homodimeric enzymes that catalyze the conversion of L-cysteine to L-alanine and sulfane sulfur via the formation of a protein-bound cysteine persulfide intermediate on a conserved cysteine residue. The enzymes are capable of donating the persulfide sulfur atoms to a variety of biosynthetic pathways for sulfur-containing biofactors, such as iron–sulfur clusters, thiamin, transfer RNA thionucleosides, biotin, and lipoic acid. The enormous advances in biochemical and structural studies of these biosynthetic pathways over the past decades provide an opportunity for detailed understanding of the nature of the excellent sulfur transfer mechanism of cysteine desulfurases.     

Biotin synthase is a pyridoxal phosphate-dependent cysteine desulfurase

Biotin synthase (BioB) is an iron-sulfur dimeric enzyme which catalyzes the last step in biotin synthesis. The reaction consists of the introduction of a sulfur atom into dethiobiotin. It is shown here that BioB displays a significant cysteine desulfurase activity, providing it with the ability to mobilize sulfur from free cysteine. This activity is dependent on pyridoxal 5'-phosphate (PLP) and dithiothreitol and proceeds through a protein-bound persulfide. Like other cysteine desulfurases, BioB binds 1 equiv of PLP. By site-directed mutagenesis, two conserved cysteines, Cys97 and Cys128, are shown to be critical for cysteine desulfuration and are good candidates as the site for a persulfide. Since biotin synthase activity is greatly increased by PLP and cysteine, even though it does not exceed 1 nmol of biotin/nmol of monomer, it is proposed that cysteine desulfuration is intimately linked to biotin synthesis. New scenarios for sulfur insertion into dethiobiotin, in which cysteine persulfides play a key role, are discussed.