Group:SMART:2010 Pingry SMART Team: Difference between revisions

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'''Design description'''
'''Design description'''


The structure of 2,5-DKGR A is a parallel alpha-beta structure consisting of eight <scene name='2010_Pingry_SMART_Team/1a80-default/2'>alpha helices(highlighted red) and eight beta strands(highlighted blue)</scene>. The alpha-beta structural motif is common among other enzymes of the aldo-keto reductase(AKR) family. Also conserved in the members of the AKR family is the residue,<scene name='2010_Pingry_SMART_Team/1a80-original/10'>Tyr50</scene> that critically functions as both the proton donor and part of the catalytic triad. The residues,<scene name='2010_Pingry_SMART_Team/1a80-original/7'>Ala47 and Trp77</scene>, are demonstrated in all AKR enzymes as well as contributors to the formation of the substrate binding pocket at the C-terminal side of the barrel. Located on an extended conformation from the outer edge of the barrel is the binding site on 2,5-DKGR A for the  
2,5-DKGR A possesses a parallel alpha-beta structural motif of the <scene name='2010_Pingry_SMART_Team/1a80-default/2'>eight alpha helices (highlighted red) and eight beta strands (highlighted blue)</scene> found in all enzymes in the aldo-keto reductase(AKR) family.  
<scene name='2010_Pingry_SMART_Team/1a80-original/12'>NADPH cofactor(shown in wireframe and colored CPK)</scene>. The NADPH cofactor is stabilized through hydrogen bonds, ionic bonds, and an aromatic pi-stacking interaction between <scene name='2010_Pingry_SMART_Team/1a80-original/2'>Trp187</scene> and the nicotinamide ring of NADPH . Although 2,5-DKGR A functions with NADPH as a cofactor, NADH is preferred for a more efficient production of vitamin C. To achieve this, mutations of the original side chains of <scene name='2010_Pingry_SMART_Team/1a80-default/1'>Lys232, Phe22, Arg238, and Ala272</scene> were conducted. Significantly, the <scene name='2010_Pingry_SMART_Team/1a80-original/8'>Lys232, Ser233, and Val234</scene> sidechain interact with the phosphate group of NADPH. In order to acommodate for the cofactor, NADH, and the absent phosphate group, these side chains have been modified in the mutant form.  
The residue <scene name='2010_Pingry_SMART_Team/1a80-original/10'>Tyr50</scene> is found at the bottom of the active-site pocket and is conserved in all members of the AKR family. The catalytic mechanism in 2,5 DKGR A is similar to aldose reductase and other members of that super family.
1. The first step involves transferring a hydride ion (H-) from NADPH to the substrate leaving an oxidized cofactor.
2. The second step involves transferring a proton (H+) to the substrate
Tyr50 is the most likely proton donor in the catalytic mechanism, making it part of the catalytic triad.
The residues, <scene name='2010_Pingry_SMART_Team/1a80-original/7'>Ala47 and Trp77</scene>, are also found in all AKR enzymes in the active-site pocket. The active site pocket of 2,5 DKGR A is significantly smaller than the active-site pocket of human aldose reductase. The bottom of the pocket is made up of residues Phe22, Asp45, Ala47, Tyr50 (mentioned above), Lys75, Leu106, Ser139, Asn140, Trp187. The top rim of the pocket is formed by non-aromatic and apolar residues Ile49, Trp77, His108, and Trp109. The C-terminal is made up of residues Ser271 to Asp278. Ala47 and Trp77 are the only residues that are conserved in all AKR’s out of all of the active site residues. The C-terminus residues are involved in the formation of hydrogen bonds with the carbohydrate substrate as well as controlling the entry and alignment of the substrate in the active site.
Located on an extended conformation from the outer edge of the barrel is the binding site on 2,5-DKGR A for the <scene name='2010_Pingry_SMART_Team/1a80-original/12'>NADPH cofactor(shown in wireframe and colored CPK)</scene>. The NADPH cofactor is stabilized through hydrogen bonds, ionic bonds, and an aromatic pi-stacking interaction between <scene name='2010_Pingry_SMART_Team/1a80-original/2'>Trp187</scene> and the nicotinamide ring of NADPH. Although 2,5-DKGR A functions with NADPH as a cofactor, NADH is preferred for a more efficient production of vitamin C. To achieve this, mutations of the original side chains of Lys232, Phe22, Arg238, and Ala272 were conducted. Significantly, the L<scene name='2010_Pingry_SMART_Team/1a80-default/1'>Lys232, Phe22, Arg238, and Ala272</scene> side chain interact with the phosphate group of NADPH. In order to accommodate for the cofactor, NADH, and the absent phosphate group, these side chains have been modified in the mutant form.




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