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The function and mechanism of AlkB - Research Paper Example

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Investigations on crystal structure and mechanism of AlkB have involved the use of both native and recombinant protein. Recombinant enzyme, entire as well as truncated form is produced through homologous expression; the former cloned in pET21b vector and expressed in BL21 (DES) competent cells; …
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The function and mechanism of AlkB
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Download file to see previous pages Iron is known to interfere in the assays due to instability of AlkB/Fe+2 complexes in aerobic conditions; a problem that is overcome by replacing iron with other metals or performing assays under anaerobic conditions. The presence of iron bound to 2-oxoglutarate in the core of the enzyme has been established through over expression and isolation of native protein. Like all other Fe(II)/2-oxoglutarate-dependent dioxygenase superfamily enzymes AlkB too has a metal center exhibiting a UV-Vis band range of 52-580nm; that in absence of DNA is a five coordinate Fe2 center and changes to six coordinate center in presence of single stranded DNA. Like other members of the superfamily, AlkB too has a core jelly roll fold that is formed of 8 beta strands at the carboxy-terminus. The catalytic domain is contained within the carboxy-terminus; however additional features for substrate specificity lie outside the catalytic domain and the jelly roll fold. At the N-terminus additional beta strands and alpha helices form a support scaffold for the catalytic domain and also the outer walls of Binding groove for DNA/RNA. In addition to these and many other structural similarities of AlkB to other members of Fe(II)/2-oxoglutarate-dependent dioxygenase superfamily; certain characteristics unique to AlkB include nucleotide binding lid, flipping mechanism motif. A common double stranded beta helix (DSBH) fold comprising of a large and a small beta sheet with iron core in between has the enzymes’ conserved residues. The first of the two distinct regions of DSBH includes a highly conserved iron binding region H131XD133XH187, wherein iron is bound to the 2-oxoglutarate in a bidentate form. The DSBH present the substrate binding site interacting exclusively with the damaged DNA/RNA strand through 2 amino terminal alpha helices and beta sheet loops that form a secondary structure called ‘lid’ (nucleotide recognition lid) over the active site. The flexible conformation of the lid allows it its amino acids (Thr51, Tyr76 and Arg161) to bind to varied alkyl groups on its nucleotide substrates through H-bonding to phosphate group in the nucleotide backbone. As a consequence of this interaction the catalytic core of the AlkB, the enzyme loses its flexibility; undergoes a conformational change that disallows oxygen to reach the active site thereby preventing the oxidation of iron. It can thus be proposed that DNA binding if occurring after iron would lead to access of oxygen to active site. DNA/RNA repair mechanism of AlkB involves oxidative demethylation of nucleotides at the site of lesion which is accomplished through hydroxylation of methylated bases through oxidative decarboxylation of 2-oxoglutarate in the enzyme core. The latter as result is converted to succinate and CO2, and methyl group is released as formaldehyde. Though the actual mechanism is yet to be established, on the basis of studies on another enzyme of the same superfamily, TauD; the probable mechanism involving an oxidative intermediate to Trp178 has been proposed. The mechanism also verified through in vitro assays on purified AlkB involves the binding of Fe2 and 2-oxoglutarate to the enzyme core followed by binding of methylated middle base to the ‘lid’. This allows oxygen to reach the iron and form nucleophillic superoxo anion (O­2-) –Fe3. This then forms a bridged peroxo-type intermediate along with 2-oxoglutarate, that through decarboxylation of 2-oxoglutarate and cleavage of O2 forms Fe4-oxygen intermediate. Coupled to oxygen cleavage is formation of succinate and CO2 from 2-oxoglutarate. The intermediate on the other hand hydoxylates the methyl group at the nucleotide forming ...Download file to see next pagesRead More
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