The function and mechanism of AlkB Research Paper

The function and mechanism of AlkB - Research Paper ExampleIron 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 branding iron with other metals or performing assays under anaerobic conditions. The charge of iron qualify to 2-oxoglutarate in the core of the enzyme has been established by dint of over expression and isolation of native protein. Like entirely 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 desoxyribonucleic acid 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 dig up that is formed of 8 important strands at the carboxy-terminus. The catalytic domain is contained within the carboxy-terminus however additional features for substratum specifi city lie outside the catalytic domain and the jelly roll twist. At the N-terminus additional beta strands and of import helices form a support scaffold for the catalytic domain and also the outer walls of Binding heat 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 take on nucleotide attach 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 amidst has the enzymes conserved residues. The first of the two distinct spheres 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 wholly with the damaged DNA/RNA strand through 2 amino terminal alpha helices and beta sheet loops that form a secondary structure c alled lid (nucleotide recognition lid) over the active site. The tractile 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 tractability undergoes a conformational change that disallows oxygen to reach the active site thereby preventing the oxidation of iron. It can thence 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 presumable 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 segmentation 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