Littérature scientifique sur le sujet « Sulfur insertase »

The lar operon in Lactobacillus plantarum encodes five Lar proteins (LarA/B/C/D/E) that collaboratively synthesize and incorporate a niacin-derived Ni-containing cofactor into LarA, an Ni-dependent lactate racemase. Previous studies have established that two molecules of LarE catalyze successive thiolation reactions by donating the sulfur atom of their exclusive cysteine residues to the substrate. However, the catalytic mechanism of this very unusual sulfur-sacrificing reaction remains elusive. In this work, we present the crystal structures of LarE in ligand-free and several ligand-bound forms, demonstrating that LarE is a member of the N-type ATP pyrophosphatase (PPase) family with a conserved N-terminal ATP PPase domain and a unique C-terminal domain harboring the putative catalytic site. Structural analysis, combined with structure-guided mutagenesis, leads us to propose a catalytic mechanism that establishes LarE as a paradigm for sulfur transfer through sacrificing its catalytic cysteine residue.

ABSTRACT Rhodobacter capsulatus xanthine dehydrogenase (XDH) is composed of two subunits, XDHA and XDHB. Immediately downstream ofxdhB, a third gene was identified, designatedxdhC, which is cotranscribed with xdhAB. Interposon mutagenesis revealed that the xdhC gene product is required for XDH activity. However, XDHC is not a subunit of active XDH, which forms an α2β2 heterotetramer inR. capsulatus. It was shown that XDHC neither is a transcriptional regulator for xdh gene expression nor influences XDH stability. To analyze the function of XDHC for XDH inR. capsulatus, inactive XDH was purified from anxdhC mutant strain. Analysis of the molybdenum cofactor content of this enzyme demonstrated that in the absence of XDHC, no molybdopterin cofactor MPT is present in the XDHAB tetramer. In contrast, absorption spectra of inactive XDH isolated from thexdhC mutant revealed the presence of iron-sulfur clusters and flavin adenine dinucleotide, demonstrating that XDHC is not required for the insertion of these cofactors. The absence of MPT from XDH isolated from an xdhC mutant indicates that XDHC either acts as a specific MPT insertase or might be a specific chaperone facilitating the insertion of MPT and/or folding of XDH during or after cofactor insertion.

ABSTRACT Bacterial nitrous oxide (N2O) reductase is the terminal oxidoreductase of a respiratory process that generates dinitrogen from N2O. To attain its functional state, the enzyme is subjected to a maturation process which involves the protein-driven synthesis of a unique copper-sulfur cluster and metallation of the binuclear CuA site in the periplasm. There are seven putative maturation factors, encoded by nosA, nosD, nosF, nosY, nosL, nosX, and sco. We wanted to determine the indispensable proteins by expressing nos genes from Pseudomonas stutzeri in the nondenitrifying organism Pseudomonas putida. An in silico study of denitrifying bacteria revealed that nosL, nosX (or a homologous gene, apbE), and sco, but not nosA, coexist consistently with the N2O reductase structural gene and other maturation genes. Nevertheless, we found that expression of only three maturation factors (periplasmic protein NosD, cytoplasmic NosF ATPase, and the six-helix integral membrane protein NosY) together with nosRZ in trans was sufficient to produce catalytically active holo-N2O reductase in the nondenitrifying background. We suggest that these obligatory factors are required for Cu-S center assembly. Using a mutational approach with P. stutzeri, we also studied NosA, the Cu-containing outer membrane protein previously thought to have Cu insertase function, and ScoP, a putative membrane-anchored chaperone for CuA metallation. Both of these were found to be dispensable elements for N2O reductase biosynthesis. Our experimental and in silico data were integrated in a model of N2O reductase maturation.

Representatives of filamentous colorless sulfur-oxidizing bacteria often dominate in sulfide biotopes, preventing the diffusion of toxic sulfide into the water column. One of the most intriguing groups is a recently described Beggiatoa leptomitoformis including strains D-401 and D-402T. Both strains have identical genes encoding enzymes which are involved in the oxidation of hydrogen sulfide and thiosulfate. Surprisingly, the B. leptomitoformis strain D-401 is not capable to grow lithotrophically in the presence of reduced sulfur compounds and to accumulate elemental sulfur inside the cells, in contrast to the D-402T strain. In general, genomes of D-401 and D-402T have an extremely high level of identity and only differ in 1 single-letter substitution, 4 single-letter indels, and 16 long inserts. Among long inserts, 14 are transposons. It was shown that in the D-401 strain, a gene coding for a sulfur globule protein was disrupted by one of the mentioned transposons. Based on comparative genomics, RT-qPCR, and HPLC-MS/MS, we can conclude that this gene plays a crucial role in the formation of the sulfur globules inside the cells, and the disruption of its function prevents lithotrophic growth of B. leptomitoformis in the presence of reduced sulfur compounds.

The regulatory gene cys-3+ controls the synthesis of a number of enzymes involved in sulfur metabolism. cys-3 mutants show a multiple loss of enzymes in different pathways of sulfur metabolism. The cys-3+ gene was isolated by transformation of an aro-9 qa-2 cys-3 inl strain with a clone bank followed by screening with the "sib selection" method. The library used (pRAL1) contained inserts of Sau3a partial digest fragments of about 9 kilobases as well as the Neurospora qa-2+ gene. Double selection for qa-2+ and cys-3+ function was carried out. The transformants obtained with the isolated cys-3+ clone show recovery of the enzyme activities associated with the cys-3 mutation (e.g., arylsulfatase and sulfate permease). Restriction fragment length polymorphism experiments confirmed the identity of the clone, mRNA studies with Northern blots show that the expression of the cys-3+ gene is inducible. In contrast to cys-3+, the cys-3 (P22) mutant gene was not expressed at a higher level under sulfur-derepressed conditions.

Two high-protein root cultures of vegetable pea mutants were received [1]. In continuation a PCR analysis of the obtained root cultures genes was carried out according [2] and the amino acid composition of the cultures protein was clarified in a dry product on the AAA 339TM device [3]. Obtained results confirmed the absence of rhizobia contamination of the cultures, which grow steadily on a hormone-free media for 5 years. PCR analysis revealed that fourrolgenesA,B,C,Dwere inserted into the genome of the root culture with genotypeafaftltl, and two —rol Candrol D— in the genome of the root culture with genotypetltl. The protein composition of the obtained cultures was represented by essential and non-essential amino acids and some others. In four inserts culture, the content of essential, ketogenic, proteinogenic and sulfur-containing amino acids prevailed by 1.5–2 times. Two inserts culture has twice as much aspartic acid and proline. Both cultures lacked tryptophan. The number of inserts determines the amino acid composition most likely.

ABSTRACT Mycobacterium tuberculosis places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in Mycobacterium smegmatis was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from M. tuberculosis were determined under strict anaerobic conditions [k cat = 1.0 (±0.1) electron consumed per second, and Km(SO3 −2) = 27 (±1) μM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (sirA, originally misannotated nirA) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed che1) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe2+ into siroheme. Deletion of che1 causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery.

Les composés soufrés, tels que la cystéine et certains cofacteurs, jouent un rôle essentiel dans les processus cellulaires. Cette thèse se concentre sur deux enzymes dépendantes d’un centre [4Fe-4S], impliquées dans le métabolisme du soufre chez l'archée anaérobie Methanococcus maripaludis : MmCyuA, une L-cystéine désulfidase, et MmLarE, une sulfurtransférase dépendante de l'ATP. La première partie porte sur MmCyuA, qui catalyse la conversion de la L-cystéine en sulfure d’hydrogène et 2-aminoacrylate, ultérieurement transformé en pyruvate et ammoniac. Les structures cristallographiques de MmCyuA, que nous avons obtenues, seule et en présence de l'inhibiteur sérine ou du produit pyruvate, sont les premières structures d'une cystéine désulfidase. Ces structures ainsi que nos résultats biochimiques et analyses spectroscopiques révèlent l’aptitude de MmCyuA à lier un cluster [4Fe-4S], indispensable à l’activité catalytique, via trois ou quatre cystéines. La structure de l'enzyme en complexe avec la sérine mime l'étape initiale de la réaction et suggère un mécanisme de désulfuration de la cystéine impliquant la formation d'un intermédiaire [4Fe-5S]. Des expériences comparatives de croissance de la souche sauvage de M. maripaludis et de la souche mutante dépourvue de l'enzyme MmCyuA soulignent l’importance de MmCyuA pour une croissance cellulaire optimale et pour permettre une croissance où la cystéine est utilisée comme unique source de soufre. Nous proposons que MmCyuA puisse transférer le sulfure lié au cluster à des accepteurs en aval des voies de biosynthèse des composés soufrés, tels que les enzymes de thiolation dépendantes d’un centre [4Fe-4S]. La deuxième partie concerne la structure et le mécanisme de MmLarE. Cette enzyme catalyse la conversion séquentielle des deux groupes carboxylates du précurseur du cofacteur de la lactate racémase en thiocarboxylates. Deux classes d’enzymes LarE existent, qui utilisent un mécanisme sacrificiel où une cystéine sert de source de soufre ou un mécanisme dépendant d’un cluster [4Fe-4S]. Nous rapportons la première structure cristallographique d'une enzyme LarE [4Fe-4S]-dépendante, sous ses formes apo (sans cluster) et holo (avec cluster). La structure de holo-MmLarE montre un cluster [4Fe- 4S], coordonné par trois cystéines seulement, avec le quatrième atome de fer lié à un ligand anionique (chlorure ou groupement phosphate). Ces structures, appuyées par nos études spectroscopiques, nous permettent de proposer un mécanisme dans lequel le cluster [4Fe-4S] lie un hydrogénosulfure, formant un intermédiaire [4Fe-5S]. Ce processus est similaire à celui des enzymes de thiolation de l'ARNt dépendantes d’un cluster [4Fe-4S]
Sulfur-containing compounds, such as cysteine and certain cofactors, play crucial roles in cellular processes. This thesis explores the sulfur metabolism in the anaerobic archaeum Methanococcus maripaludis, focusing on two [4Fe-4S]-dependent enzymes: L-cysteine desulfidase MmCyuA and ATP-dependent sulfur insertase MmLarE. The first part focuses on MmCyuA, which catalyzes the decomposition of L-cysteine into hydrogenosulfide and 2-aminoacrylate, subsequently converted into pyruvate and ammonia. The crystal structures of MmCyuA that we obtained, alone and in the presence of the serine inhibitor or the pyruvate product, are the first structures of a cysteine desulfidase. These structures, together with our biochemical results and spectroscopic analysis, reveal the capacity of MmCyuA to bind a [4Fe-4S] cluster, required for activity, using three or four cysteines. The structure of the enzyme in complex with serine mimics the initial step of the reaction and suggest a desulfuration mechanism for cysteine that involves the formation of a [4Fe-5S] intermediate. Comparative growth experiments between wild-type and CyuAdeficient M. maripaludis strains highlight the important role of MmCyuA for optimal growth and to enables growth using cysteine as the sole sulfur source. We propose that MmCyuA could transfer the cluster-bound sulfide to downstream acceptors, along the biosynthetic pathways of sulfurated compounds, such as [4Fe-4S]-dependent thiolation enzymes. The second part details the structure and mechanism of MmLarE. This enzyme catalyzes the sequential conversion of the two carboxylate groups of the precursor of the lactate racemase cofactor into thiocarboxylates. Two classes of LarE enzymes exist, using a sacrificial mechanism, in which a cysteine serves as the sulfur source, or a [4Fe-4S] cluster-dependent mechanism. We present the first crystal structure of a [4Fe-4S]-dependent LarE enzyme, in both its apo (without cluster) and holo (with cluster) forms. The crystal structure of holo-MmLarE reveals a [4Fe-4S] cluster coordinated by three cysteines only, with the fourth iron atom bound to an anionic ligand (chloride or phosphate group). These structures, along with our spectroscopic studies, support a mechanism in which the [4Fe-4S] cluster binds a hydrogenosulfide ligand, forming a [4Fe-5S]