Banta:AKR: Difference between revisions
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''' | '''Engineering Dehydrogenase Activity''' | ||
Dehydrogenase enzymes are commonly used in a wide range of biotechnology applications, including chemical synthesis and bioelectrocatalysis. Aldo-keto reductases (AKRs) are a family of enzymes that catalyze a wide range of redox reactions with a broad array of substrates, and are an attractive family of enzymes for protein engineering efforts. They are monomeric and they fold into the well-known TIM-like alpha/beta structure. The cofactor binding pocket and the active site are highly conserved within the superfamily, while substrate specificity is tailored through three substrate binding loops located above the active site. | Dehydrogenase enzymes are commonly used in a wide range of biotechnology applications, including chemical synthesis and bioelectrocatalysis. Aldo-keto reductases (AKRs) are a family of enzymes that catalyze a wide range of redox reactions with a broad array of substrates, and are an attractive family of enzymes for protein engineering efforts. They are monomeric and they fold into the well-known TIM-like alpha/beta structure. The cofactor binding pocket and the active site are highly conserved within the superfamily, while substrate specificity is tailored through three substrate binding loops located above the active site. | ||
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More recently we have been exploring the cofactor and substrate specificity of the AhdD enzyme which is an AKR from the hyperthermophilic archeon Pyrococcus furiosus. This enzyme natively uses NAD(H) as its preferred cofactor and the mutations used to alter cofactor specificity in 2,5-DKGR were used to similarly broaden the cofactor specificity of AdhD. The mutant AdhD is also able to use biomimetic cofactors, and we have used this mutant to create an enzymatic biofuel cell using an biomimetic cofactor for redox cycling. We have also explored swapping the substrate-binding loops in AdhD to alter its substrate specificity in addition to its cofactor specificity. AdhD is a valuable enzymatic scaffold for engineering enzymes with high thermostability. | More recently we have been exploring the cofactor and substrate specificity of the AhdD enzyme which is an AKR from the hyperthermophilic archeon Pyrococcus furiosus. This enzyme natively uses NAD(H) as its preferred cofactor and the mutations used to alter cofactor specificity in 2,5-DKGR were used to similarly broaden the cofactor specificity of AdhD. The mutant AdhD is also able to use biomimetic cofactors, and we have used this mutant to create an enzymatic biofuel cell using an biomimetic cofactor for redox cycling. We have also explored swapping the substrate-binding loops in AdhD to alter its substrate specificity in addition to its cofactor specificity. AdhD is a valuable enzymatic scaffold for engineering enzymes with high thermostability. | ||
To further demonstrate the utility of the AdhD scaffold, we are now working to engineering the enzyme to become a binding molecule, like and antibody. We will take advantage of the high levels of production and extreme thermostability of the protein and we are working to convert it into a biomolecular recognition element for explosives detection. | |||
Revision as of 12:57, 17 July 2014
Banta Lab
Protein and Metabolic Engineering
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Engineering Dehydrogenase Activity
Dehydrogenase enzymes are commonly used in a wide range of biotechnology applications, including chemical synthesis and bioelectrocatalysis. Aldo-keto reductases (AKRs) are a family of enzymes that catalyze a wide range of redox reactions with a broad array of substrates, and are an attractive family of enzymes for protein engineering efforts. They are monomeric and they fold into the well-known TIM-like alpha/beta structure. The cofactor binding pocket and the active site are highly conserved within the superfamily, while substrate specificity is tailored through three substrate binding loops located above the active site.
We have rationally altered the cofactor specificity of a bacterial AKR, the 2,5-diketo-D-gluconic acid reductase (2,5-DKGR) which is an important enzyme for the enzymatic production of vitamin C. Both site-directed mutations as well as combinatorial approaches were used. The native enzyme is strongly NADP(H)-dependent and through this protein engineering approach a new mutant was created that retained its NADP(H) activity while gaining activity with NAD(H) that is close to the wild type activity with NADP(H).
More recently we have been exploring the cofactor and substrate specificity of the AhdD enzyme which is an AKR from the hyperthermophilic archeon Pyrococcus furiosus. This enzyme natively uses NAD(H) as its preferred cofactor and the mutations used to alter cofactor specificity in 2,5-DKGR were used to similarly broaden the cofactor specificity of AdhD. The mutant AdhD is also able to use biomimetic cofactors, and we have used this mutant to create an enzymatic biofuel cell using an biomimetic cofactor for redox cycling. We have also explored swapping the substrate-binding loops in AdhD to alter its substrate specificity in addition to its cofactor specificity. AdhD is a valuable enzymatic scaffold for engineering enzymes with high thermostability.
To further demonstrate the utility of the AdhD scaffold, we are now working to engineering the enzyme to become a binding molecule, like and antibody. We will take advantage of the high levels of production and extreme thermostability of the protein and we are working to convert it into a biomolecular recognition element for explosives detection.
Related Publications
- Campbell E, Wheeldon IR, and Banta S. Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior. Biotechnol Bioeng. 2010 Dec 1;107(5):763-74. DOI:10.1002/bit.22869 |
- Wheeldon IR, Campbell E, and Banta S. A chimeric fusion protein engineered with disparate functionalities-enzymatic activity and self-assembly. J Mol Biol. 2009 Sep 11;392(1):129-42. DOI:10.1016/j.jmb.2009.06.075 |
- Sanli G, Banta S, Anderson S, and Blaber M. Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase. Protein Sci. 2004 Feb;13(2):504-12. DOI:10.1110/ps.03450704 |
- Banta S, Boston M, Jarnagin A, and Anderson S. Mathematical modeling of in vitro enzymatic production of 2-Keto-L-gulonic acid using NAD(H) or NADP(H) as cofactors. Metab Eng. 2002 Oct;4(4):273-84. DOI:10.1006/mben.2002.0231 |
- Banta S and Anderson S. Verification of a novel NADH-binding motif: combinatorial mutagenesis of three amino acids in the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase. J Mol Evol. 2002 Dec;55(6):623-31. DOI:10.1007/s00239-002-2345-x |
- Banta S, Swanson BA, Wu S, Jarnagin A, and Anderson S. Optimizing an artificial metabolic pathway: engineering the cofactor specificity of Corynebacterium 2,5-diketo-D-gluconic acid reductase for use in vitamin C biosynthesis. Biochemistry. 2002 May 21;41(20):6226-36. DOI:10.1021/bi015987b |
- Banta S, Swanson BA, Wu S, Jarnagin A, and Anderson S. Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A. Protein Eng. 2002 Feb;15(2):131-40. DOI:10.1093/protein/15.2.131 |
