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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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?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; while the latter cloned in pET30a vector and expressed in BL21 competent cells. 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 formaldehyde though an unstable intermediate. The nucleotide binding subdomain of AlkB, in contrast to other enzymes of the superfamily binds nonspecifically to DNA/RNA methylated trinucleotiedes. It is also responsible for the base flipping mechanism utilized by AlkB for demethylation of bases. The interaction between nucleotide and AlkB being weak; it is difficult to study the AlkB base flipping mechanism. However to overcome this obstacle, a site specific covalent disulfide crosslink between DNA and enzyme has been used that was made possible by replacing with cysteine, one of the residues at the metal binding site in the enzyme. Studies on the base flipping mechanism thus made possible revealed that the methylated base is flipped out from the DNA/RNA and is lodged in the enzyme catalytic site. To make this possible, the enzyme kinks the damaged DNA/RNA at the site of lesion and brings together the flanking bases so that they come to lay one on top of other, resulting in the removal of methylated damaged base along with its pair in complementary strand. The adjacent bases undergo conformational changes in the process involving inversion of one of the sugar rings. The inverted ring is supported by interaction with the enzyme residues Thr51, Pro52 and Gly53 through H-bonds, along with many simultaneous interactions between the bases and the enzyme resulting in formation of a compressed DNA structure. The eliminated nucleotide is stacked above Trp69. Simultaneous removal of complementary base in double stranded DNA involves significant rearrangements along with much higher energy expenditure compared to single stranded DNA. This accounts for the preference of the base flipping mechanism for latter. Formation of AlkB-Succinate complex from AlkB-oxoglutarate complex during the process involves significant changes in the enzyme dynamics that can be responsible for the release of products from the enzyme. The oxidative decarboxylation of 2-oxoglutarate to succinate loosens its interaction with the large beta sheet of the enzyme since the Asn120 of AlkB that forms hydrogen bond with 2-oxoglutarate, is not able to do so with succinate. This loss of contact between enzyme and succinate separates the two sheets of DSBH, hence the AlkB/succinate complex exhibits lesser affinity for DNA. Thus the conformational change accompanying due to conversion of 2-oxoglutarate to succinate may be responsible for release of unmethylated DNA from the enzyme binding site. Read More
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