Group:SMART:2010 Pingry SMART Team
Modifying 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)
Design description
The conformation of 2.5-DKGR A is a parallel alpha/beta barrel of eight alpha helices(highlighted red) and eight beta strands(highlighted blue). Notably, the alpha-beta 8 structure is common among other enzymes of the aldo-keto reductase family. Red and blue highlight the (alpha/beta)8 barrel structure typical of AKR's.
Cofactor (NADPH) shown in wireframe and colored CPK.
(Lys232, Phe22, Arg238, Ala272).
Other residues highlighted by displaying sidechain:
Trp187; pi-stacking interaction with cofactor that stabilizes the reaction
Lys232, Ser233, Val234; interacts with phosphate group of NADPH. These are changed in the mutant form in order to accommodate for the cofactor NADH.
Not shown:
Ala47 and Trp77; two residues conserved in all AKR proteins.
Tyr50; Proton donor in AKR and part of catalytic triad that is conserved in all AKR proteins.
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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 eight alpha helices (highlighted red) and eight beta sheets (highlighted blue). 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 (Lys232Gly, Phe22Tyr, Arg238His, Ala272Gly).
Lys232Gly mutation is important because Gly has no sidechain so there is nothing to interact with the absent phosphate group.
Phe22Tyr mutation reduces the Km for both NADPH and NADH.
Arg238His mutation forms a pi-stacking interaction to stabilize the AKR with the cofactor.
Ala272Gly mutation 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:
Trp187; pi-stacking interaction with the nicotinamide ring of the cofactor that stabilizes the reaction.
Not shown:
Ala47 and Trp77; residues conserved in AKR's
Tyr50; Proton donor in AKR and part of the catalytic triad that is conserved in all AKR's
Proteopedia Page Contributors and Editors (what is this?)
Tommie Hata, Ed Xiao, Dylan Sun, Mai-Lee Picard, Connie Wang, Doug Ober, Caryn Ha, Florence Ma, David Sukhin, Ed Kong, Alexander Berchansky
