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Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior.
Biotechnol Bioeng. 2010 Dec 01; 107(5):763-74.BB

Abstract

Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site-directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type. Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2'-phosphate of the cofactor. Pre-steady-state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2'-phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex.

Authors+Show Affiliations

Department of Chemical Engineering, Columbia University, 500 West 120th Street, New York, New York 10027, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

20632378

Citation

Campbell, Elliot, et al. "Broadening the Cofactor Specificity of a Thermostable Alcohol Dehydrogenase Using Rational Protein Design Introduces Novel Kinetic Transient Behavior." Biotechnology and Bioengineering, vol. 107, no. 5, 2010, pp. 763-74.
Campbell E, Wheeldon IR, Banta S. Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior. Biotechnol Bioeng. 2010;107(5):763-74.
Campbell, E., Wheeldon, I. R., & Banta, S. (2010). Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior. Biotechnology and Bioengineering, 107(5), 763-74. https://doi.org/10.1002/bit.22869
Campbell E, Wheeldon IR, 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. PubMed PMID: 20632378.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior. AU - Campbell,Elliot, AU - Wheeldon,Ian R, AU - Banta,Scott, PY - 2010/7/16/entrez PY - 2010/7/16/pubmed PY - 2011/2/1/medline SP - 763 EP - 74 JF - Biotechnology and bioengineering JO - Biotechnol. Bioeng. VL - 107 IS - 5 N2 - Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site-directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type. Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2'-phosphate of the cofactor. Pre-steady-state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2'-phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex. SN - 1097-0290 UR - https://www.unboundmedicine.com/medline/citation/20632378/Broadening_the_cofactor_specificity_of_a_thermostable_alcohol_dehydrogenase_using_rational_protein_design_introduces_novel_kinetic_transient_behavior_ L2 - https://doi.org/10.1002/bit.22869 DB - PRIME DP - Unbound Medicine ER -