Group:SMART:2010 Pingry SMART Team: Difference between revisions
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'''Design description''' | '''Design description''' | ||
2,5-DKGR A possesses a parallel alpha-beta structure motif of eight <scene name='2010_Pingry_SMART_Team/1a80-default/2'>alpha helices(highlighted red) and eight beta strands(highlighted blue)</scene>found in other enzymes of the aldo-keto reductase(AKR) family. Also conserved in members of the AKR family is the residue,<scene name='2010_Pingry_SMART_Team/1a80-original/10'>Tyr50</scene>, that 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 | 2,5-DKGR A possesses a parallel alpha-beta structure motif of eight <scene name='2010_Pingry_SMART_Team/1a80-default/2'>alpha helices(highlighted red) and eight beta strands(highlighted blue)</scene>found in other enzymes of the aldo-keto reductase(AKR) family. Also conserved in members of the AKR family is the residue,<scene name='2010_Pingry_SMART_Team/1a80-original/10'>Tyr50</scene>, that 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. Located at the C-terminal side of the barrel is the active site for the | ||
<scene name='2010_Pingry_SMART_Team/1a80-original/12'>NADPH cofactor(shown in wireframe and colored CPK)</scene> to bind to 2.5-DKGR A. 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, 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. | <scene name='2010_Pingry_SMART_Team/1a80-original/12'>NADPH cofactor(shown in wireframe and colored CPK)</scene> to bind to 2.5-DKGR A. 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, 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. | ||
Revision as of 09:23, 1 February 2010
Modifying cofactor specificity, 2,5-diketo-d-gluconic acid reductaseModifying cofactor specificity, 2,5-diketo-d-gluconic acid reductase
2,5 Diketo-D-Gluconic acid reductase is found in corynebacterium and is part of the Aldo Keto Reductase family of enzymes. It exists in two variants: DKGR A and DKGR B; however, due to the higher thermal stability level of DKGR A, it has been chosen for mutation of cofactor specificity. 2,5 DKGR is an important enzyme in the production of vitamin C, one of the most important chemicals manufactured in the world. 2,5 DKGR does this by catalyzing the reduction of 2,5-diketo-D-gluconic acid (2,5 DKG) to 2-Keto-L-gluconic acid (2-KLG); a precursor to vitamin C. It is commercially less expensive to use NADH as a cofactor (as opposed to NADPH) and the catalyzation of 2,5 DKG into 2-KLG as well as being more abundant.
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PDB ID: 1a80, 2,5-diketo-d-gluconic acid reductase with NADPH (wild-type)PDB ID: 1a80, 2,5-diketo-d-gluconic acid reductase with NADPH (wild-type)
Design description
2,5-DKGR A possesses a parallel alpha-beta structure motif of eight found in other enzymes of the aldo-keto reductase(AKR) family. Also conserved in members of the AKR family is the residue,, that functions as both the proton donor and part of the catalytic triad. The residues,, are demonstrated in all AKR enzymes as well as contributors to the formation of the substrate binding pocket. Located at the C-terminal side of the barrel is the active site for the to bind to 2.5-DKGR A. The NADPH cofactor is stabilized through hydrogen bonds, ionic bonds, and an aromatic pi-stacking interaction between 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 were conducted. Significantly, the 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.
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PDB ID: 1m9h, Mutant 2,5-diketo-d-gluconic acid reductase with NADH (mutant)PDB ID: 1m9h, Mutant 2,5-diketo-d-gluconic acid reductase with NADH (mutant)
Design description
The conformation of 2,5-DKGR (diketo-d-gluconic acid reductase)is a parallel alpha/beta barrel of Notably, the alpha/beta 8 barrel structure is demonstrated by other enzymes of the aldo-keto reductase family.
The backbone of the four residues changed between WT and NADP-binding mutant are colored orange
is important because Gly has no sidechain so there is nothing to interact with the absent phosphate group.
reduces the Km for both NADPH and NADH.
forms a pi-stacking interaction to stabilize the AKR with the cofactor.
improves the kinetic properties by making it easier for the substrate to bind with the substrate or by improving the kinetics of cofactor binding and release.
Other residues highlighted by displaying sidechain:
pi-stacking interaction with the nicotinamide ring of the cofactor that stabilizes the reaction.
Not shown:
residues conserved in AKR's
Proton donor in AKR and part of the catalytic triad that is conserved in all AKR's