Parsing redox potentials of five ferredoxins found within Thermotoga maritima

Most organisms contain multiple soluble protein‐based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron–sulfur clusters. The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the “radical SAM,” or S‐adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron–sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe₄S₄]¹⁺ oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side‐by‐side fashion for the first time. While a single [Fe₄S₄]‐cluster bearing Fd (TM0927) has a potential of ?420 mV, the other four 2x[Fe₄S₄]‐bearing Fds (TM1175, TM1289, TM1533, and TM1815) have potentials that vary significantly, including cases where the two clusters of the same Fd are essentially coincident (e.g., TM1175) and those where the potentials are well separate (TM1815).     

Molecular architecture of KedS8, a sugar N‐methyltransferase from Streptoalloteichus sp. ATCC 53650

Kedarcidin, produced by Streptoalloteichus sp. ATCC 53650, is a fascinating chromoprotein of 114 amino acid residues that displays both antibiotic and anticancer activity. The chromophore responsible for its chemotherapeutic activity is an ansa‐bridged enediyne with two attached sugars, l ‐mycarose, and l ‐kedarosamine. The biosynthesis of l ‐kedarosamine, a highly unusual trideoxysugar, is beginning to be revealed through bioinformatics approaches. One of the enzymes putatively involved in the production of this carbohydrate is referred to as KedS8. It has been proposed that KedS8 is an N‐methyltransferase that utilizes S‐adenosylmethionine as the methyl donor and a dTDP‐linked C‐4′ amino sugar as the substrate. Here we describe the three‐dimensional architecture of KedS8 in complex with S‐adenosylhomocysteine. The structure was solved to 2.0 Å resolution and refined to an overall R‐factor of 17.1%. Unlike that observed for other sugar N‐methyltransferases, KedS8 adopts a novel tetrameric quaternary structure due to the swapping of β‐strands at the N‐termini of its subunits. The structure presented here represents the first example of an N‐methyltransferase that functions on C‐4′ rather than C‐3′ amino sugars.     

A Glycine N-methyltransferase Knockout Mouse Model for Humans with Deficiency of this Enzyme

Three human cases having mutations in the glycine N-methyltransferase (GNMT) gene have been reported. This enzyme transfers a methyl group from S-adenosylmethionine (SAM) to glycine to form S-adenosylhomocysteine (SAH) and N-methylglycine (sarcosine) and is believed to be involved in the regulation of methylation. All three cases have mild liver disease but they seem otherwise unaffected. To study this further, gnmt deficient mice were generated for the first time. This resulted in the complete absence of GNMT protein and its activity in livers of homozygous mice. Compared to WT animals the absence of GNMT resulted in up to a 7-fold increase of free methionine and up to a 35-fold increase of SAM. The amount of SAH was significantly decreased (3 fold) in the homozygotes compared to WT. The ratio of SAM/SAH increased from 3 in WT to 300 in livers of homozygous transgenic mice. This suggests a possible significant change in methylation in the liver and other organs where GNMT is expressed.     

Identification of a Protein that Interacts with LeATL6 Ubiquitin‐protein Ligase E3 Upregulated in Tomato Treated with Elicitin‐Like Cell Wall Proteins of Pythium oligandrum

The expression of LeATL6, which encodes RING‐H2 zinc finger ubiquitin‐protein ligase E3, is highly induced in tomato roots treated with the elicitin‐like cell wall protein fraction (CWP) from the non‐pathogenic oomycete Pythium oligandrum, which enhances resistance to pathogens through a jasmonic acid (JA)‐dependent signalling pathway. In this study, the role of LeATL6 for CWP‐induced defence response was further analysed. To screen the putative target protein of LeATL6 for the CWP‐induced defence mechanism in tomato, we used a yeast two‐hybrid system to screen five clones encoding a protein that interacts with LeATL6. Four clones had a function associated with the ubiquitin‐proteasome system. Another positive clone encoded a protein sharing homology with S‐adenosylmethionine decarboxylase (SAMDC). In CWP‐treated tomato roots, SAMDC activity was clearly suppressed. Thus, the interaction of SAMDC with LeATL6 and the decreased SAMDC activity may be associated with JA‐dependent induced resistance in tomato treated with P. oligandrum.     

Ferredoxins as interchangeable redox components in support of MiaB,a radical S‐adenosylmethionine methylthiotransferase

MiaB is a member of the methylthiotransferase subclass of the radical S‐adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N⁶‐isopentenyl (i⁶A) group found at position 37 in several tRNAs to afford 2‐methylthio‐N⁶‐(isopentenyl)adenosine (ms²i⁶A). MiaB uses a reduced [4Fe–4S]⁺ cluster to catalyze a reductive cleavage of SAM to generate a 5′‐deoxyadenosyl 5′‐radical (5′‐dA?)—a required intermediate in its reaction—as well as an additional [4Fe–4S]²⁺ auxiliary cluster. In Escherichia coli and many other organisms, re‐reduction of the [4Fe–4S]²⁺ cluster to the [4Fe–4S]⁺ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin‐NADP⁺ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5′‐dA? and ms²i⁶A production, indicating that only one hydrogen atom abstraction is required for the reaction.     

Antioxidant Properties of S-Adenosyl-l-Methionine: A Proposed Addition to Organ Storage Fluids

Glutathione (GSH) depletion adversely affects the survival of organ grafts. Supplementation of commercial organ preservation solutions with GSH is complicated by the ease of oxidation of its thiol group and its ability to act as a pro-oxidant under certain conditions. Alternative sulphur-containing compounds such as S-adenosyl-

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-methionine (SAM) can reduce ischaemia-reperfusion injury, possibly by acting as glutathione precursors, and are effective when added to preservation solutions. Although the antioxidant properties of GSH are known in some detail, there is little information on the ability of SAM to interact directly with reactive oxygen species (ROS) produced during ischaemia-reperfusion injury. This work compares the interaction of SAM and GSH with several ROS which may be formed during ischaemia-reperfusion. In a variety of lipid peroxidation systems, SAM and GSH had little effect except at high concentrations (5 mM) where they became pro-oxidant. Scavenging of O₂^˙− by both species was slow. SAM was less effective than GSH at preventing damage by peroxynitrite or HOCl. In contrast, SAM was more effective than GSH in scavenging hydroxyl radicals (^˙OH) and in chelating iron ions to inhibit ^˙OH generation. Unlike GSH, SAM did not stimulate ^˙OH formation at low concentrations. The beneficial effects of SAM in preservation solutions could therefore include direct radical scavenging as well as acting as a precursor for intracellular GSH.     

Polyamine depletion by ODC-AdoMetDC antisense adenovirus impairs human colorectal cancer growth and invasion in vitro and in vivo

BACKGROUND: Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Polyamine concentrations are elevated in colorectal cancer. Depletion of polyamine content in colorectal cancer by chemotherapy is related to tumor regression and impaired tumorigenicity. The current study evaluates the therapeutic effects of antisense ODC and AdoMetDC sequences on colorectal cancer in vitro and in vivo. METHODS: Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad-ODC-AdoMetDCas). The human colon cancer cell lines, HT-29 and Caco-2, were infected with Ad-ODC-AdoMetDCas as well as with control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were performed in order to assess properties of tumor growth and invasiveness. Furthermore, the antitumor effects of Ad-ODC-AdoMetDCas were also evaluated in vivo in a nude mouse tumor model. RESULTS: Our study demonstrated that adenovirus-mediated ODC and AdoMetDC antisense expression inhibits tumor cell growth through a blockade of the polyamine synthesis pathway. This inhibitory effect cannot be reversed by the administration of putrescine. Tumor cells were arrested at the G1 phase of the cell cycle after gene transfer and had reduced invasiveness. The adenovirus also induced tumor regression in established tumors in nude mice. CONCLUSIONS: Our study suggests that Ad-ODC-AdoMetDCas has antitumor activity and therapeutic potential for the treatment of colorectal cancer.