Enzyme catalyzed formation of radicals from S-adenosylmethionine and inhibition of enzyme activity by the cleavage products

A large superfamily of enzymes have been identified that make use of radical intermediates derived by reductive cleavage of S-adenosylmethionine. The primary nature of the radical intermediates makes them highly reactive and potent oxidants. They are used to initiate biotransformations by hydrogen atom abstraction, a process that allows a particularly diverse range of substrates to be functionalized, including substrates with relatively inert chemical structures. In the first part of this review, we discuss the evidence supporting the mechanism of radical formation from S-adenosylmethionine. In the second part of the review, we examine the potential of reaction products arising from S-adenosylmethionine to cause product inhibition. The effects of this product inhibition on kinetic studies of ‘radical S-adenosylmethionine’ enzymes are discussed and strategies to overcome these issues are reviewed. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis

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Four human thiopurine s-methyltransferase alleles severely affect protein structure and dynamics

Thiopurine S-methyltransferase (TPMT) metabolizes cytotoxic thiopurine drugs used in the treatment of leukemia and inflammatory bowel disease. TPMT is a major pharmacogenomic target with 23 alleles identified to date. Several of these alleles cause rapid protein degradation and/or aggregation, making it extremely difficult to study the structural impact of the TPMT polymorphisms experimentally. We, therefore, have performed multiple molecular dynamics simulations of the four most common alleles [TPMT*2 (A80P), *3A (A154T/Y240C), *3B (A154T) and *3C (Y240C)] to investigate the molecular mechanism of TPMT inactivation at an atomic level. The A80P polymorphism in TPMT*2 disrupts helix α3 bordering the active site, which breaks several salt-bridge interactions and opens up a large cleft in the protein. The A154T polymorphism is located within the co-substrate binding site. The larger threonine alters the packing of substrate-binding residues (P68, L69, Y166), increasing the solvent exposure of the polymorphic site. This packing rearrangement may account for the complete lack of activity in the A154T mutant. The Y240C polymorphism is located in β-strand 9, distant from the active site. Side-chain contacts between residue 240 and helix α8 are lost in TPMT*3C. Residues 154 and 240 in TPMT*3A are connected through a hydrogen-bonding network. The dual polymorphisms result in a flattened, slightly distorted protein structure and an increase in the thiopurine-binding site solvent accessibility. The two variants that undergo the most rapid degradation in vivo, TPMT*2 and *3A, are also the most deformed in the simulations.     

CCL20在脑卒中的发病机制及临床意义

CCL20(C-C chemokine ligand 20)是CC亚族的趋化因子,曾被称为巨噬细胞炎症蛋白3α(MIP-3α)、肝脏活化调节趋化因子(LARC)和Exodus-1,与其唯一受体CC趋化因子受体6(CC chemokine receptor 6,CCR6)特异性结合,调节细胞的激活、趋化和迁移,参与形态发生、组织稳态、固有和获得性免疫以及许多疾病的炎症病理过程。在中枢神经中,CCL20与CCR6结合后激活小胶质细胞,其可应激相关性蛋白,形成持续性神经毒性级联反应,从而进一步损害周围神经元和脑组织,进而引起一系列的神经功能损伤症状。目前有关CCL20在脑卒中的日益引起人们的关注,深入研究其在脑卒中的作用,有可能为进一步揭示脑卒中的发病机制并为寻求有效的防治措施提供重要的理论基础。本文概述了CCL20的作用,重点阐述了其在脑卒中的作用。     

Crystal structure and biochemical studies of Brucella melitensis 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase

The prokaryotic 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the irreversible cleavage of the glycosidic bond in 5′-methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH), a process that plays a key role in several metabolic pathways. Its absence in all mammalian species has implicated this enzyme as a promising target for antimicrobial drug design. Here, we report the crystal structure of BmMTAN in complex with its product adenine at a resolution of 2.6 Å determined by single-wavelength anomalous dispersion method. 11 key residues were mutated for kinetic characterization. Mutations of Tyr134 and Met144 resulted in the largest overall increase in Km, whereas mutagenesis of residues Glu18, Glu145 and Asp168 completely abolished activity. Glu145 and Asp168 were identified as active site residues essential for catalysis. The catalytic mechanism and implications of this structure for broad-based antibiotic design are discussed.     

Inhibition of Rho kinase by hydroxyfasudil attenuates brain edema after subarachnoid hemorrhage in rats

The blood-brain barrier (BBB) disruption and brain edema are important pathophysiologies of early brain injury after subarachnoid hemorrhage (SAH). This study is to evaluate whether Rho kinase (Rock) enhances BBB permeability via disruption of tight junction proteins during early brain injury. Adult male rats were assigned to five groups; Sham-operated, SAH treated with saline, a Rock inhibitor hydroxyfasudil (HF) (10 mg/kg) treatment at 0.5 h after SAH, HF treatment at 0.5 and 6 h (10 mg/kg, each) after SAH, and another Rock inhibitor Y27632 (10 mg/kg) treatment at 0.5 h after SAH. The perforation model of SAH was performed and neurological score and brain water content were evaluated 24 and 72 h after surgery. Evans blue extravasation, Rock activity assay, and western blotting analyses were evaluated 24 h after surgery. Treatment of HF significantly improved neurological scores 24 h after SAH. Single treatment with HF and Y27632, and two treatments with HF reduced brain water content in the ipsilateral hemisphere. HF reduced Evans blue extravasation in the ipsilateral hemisphere after SAH. Rock activity increased 24 h after SAH, and HF reversed the activity. SAH significantly decreased the levels of tight junction proteins, occludin and zonula occludens-1 (ZO-1), and HF preserved the levels of occluding and ZO-1 in ipsilateral hemisphere. In conclusion, HF attenuated BBB permeability after SAH, possibly by protection of tight junction proteins.     

Biochemical and structural characterization of 5′-methylthioadenosine nucleosidases from Arabidopsis thaliana

5′-Methylthioadenosine (MTA) and S-adenosylhomocysteine (SAH) are important metabolites in all living organisms. Two similar nucleosidases for hydrolyzing MTA in Arabidopsis thaliana (AtMTAN1 and AtMTAN2) exist, but only AtMTAN2 shows markedly broad substrate specificity for hydrolysis of SAH. To examine the biochemical characteristics of AtMTAN2, it was over-expressed in Escherichia coli and purified to homogeneity. Spectroscopic assays confirm AtMTAN2 catalyzes MTA as well as SAH hydrolysis, compared to AtMTAN1 which only hydrolyzes MTA. In addition, crystal structure of the AtMTAN2 enzyme in complex with, adenine was determined at 2.9 Å resolution. Finally, a structural comparison of AtMTAN2 performed with previously determined structures of AtMTAN1 and an E. coli homolog provides clues for the substrate specificity of MTA nucleosidases in A. thaliana.     

The antiviral protein viperin is a radical SAM enzyme

Viperin, an interferon-inducible antiviral protein, is shown to bind an iron-sulfur cluster, based on iron analysis as well as UV-Vis and electron paramagnetic resonance spectroscopic data. The reduced protein contains a [4Fe-4S]¹⁺ cluster whose g-values are altered upon addition of S-adenosylmethionine (SAM), consistent with SAM coordination to the cluster. Incubation of reduced viperin with SAM results in reductive cleavage of SAM to produce 5′-deoxyadenosine (5′-dAdo), a reaction characteristic of the radical SAM superfamily. The 5′-dAdo cleavage product was identified by a combination of HPLC and mass spectrometry analysis.     

Interaction of the tylosin-resistance methyltransferase RlmA II at its rRNA target differs from the orthologue RlmA I

RlmA^II methylates the N1-position of nucleotide G748 in hairpin 35 of 23 S rRNA. The resultant methyl group extends into the peptide channel of the 50 S ribosomal subunit and confers resistance to tylosin and other mycinosylated macrolide antibiotics. Methylation at G748 occurs in several groups of Gram-positive bacteria, including the tylosin-producer Streptomyces fradiae and the pathogen Streptococcus pneumoniae. Recombinant S. pneumoniae RlmA^II was purified and shown to retain its activity and specificity in vitro when tested on unmethylated 23 S rRNA substrates. RlmA^II makes multiple footprint contacts with nucleotides in stem-loops 33, 34 and 35, and does not interact elsewhere in the rRNA. Binding of RlmA^II to the rRNA is dependent on the cofactor S-adenosylmethionine (or S-adenosylhomocysteine). RlmA^II interacts with the same rRNA region as the orthologous enzyme RlmA^I that methylates at nucleotide G745. Differences in nucleotide contacts within hairpin 35 indicate how the two methyltransferases recognize their distinct targets.     

Structural and functional insights into O-methyltransferase from Bacillus cereus

The specific substrates, mechanisms, and structures of the bacterial O-methyltransferases (OMTs) are not as well characterized as those of other OMTs. Recent studies have suggested that bacterial OMTs catalyze regiospecific reactions that might be used to produce novel compounds. In this study, we investigated the structural and functional features of an OMT from Bacillus cereus (BcOMT2). This enzyme catalyzes the O-methylation of flavonoids in vitro in an S-adenosylmethionine-dependent and regiospecific manner. We solved the crystal structures of the BcOMT2 apoenzyme and the BcOMT2-S-adenosylhomocysteine (SAH) co-complex at resolutions of 1.8 and 1.2 Å, respectively. These structures reveal that the overall structure of dimeric BcOMT2 is similar to that of the canonical OMT but that BcOMT2 also has a unique N-terminal helical region that is responsible for dimerization. The binding of SAH causes both local and remote conformational changes in the dimer interface that stabilize the dimerization of BcOMT2. SAH binding also causes ordering of residues Glu171 to Gly186, which are disordered in the apoenzyme structure and are known determinants of substrate specificity, and thus contributes to formation of the substrate binding pocket. Our structural analysis indicated a resemblance between the active site of BcOMT2 and that of metal-dependent OMTs. Using mutational analysis, we confirmed that BcOMT2 is a Mg²⁺-dependent OMT. These results provide structural and functional insights into the dimerization mechanism and substrate specificity of BcOMT2.