Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior

Biotechnology and Bioengineering
Elliot CampbellScott Banta

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 indep...Continue Reading

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Citations

Jan 10, 2012·Chemical Communications : Chem Comm·Elliot CampbellScott Banta
Mar 27, 2012·Trends in Biotechnology·Y-H Percival Zhang, Wei-Dong Huang
Jul 12, 2011·Current Opinion in Biotechnology·Haruyuki AtomiTamotsu Kanai
May 24, 2012·Protein Science : a Publication of the Protein Society·Flora FelsovalyiScott Banta
Feb 16, 2013·Current Opinion in Chemical Engineering·Andrew D Frock, Robert M Kelly
Oct 17, 2014·Biotechnology Journal·Quentin M DudleyMichael C Jewett
Nov 15, 2011·Biotechnology Advances·Yan Ni, Jian-He Xu
Jan 1, 2012·Computational and Structural Biotechnology Journal·Laura R JarboeLonnie O Ingram
Apr 16, 2013·BioTechniques·Oren ShurScott Banta
Nov 23, 2017·Chembiochem : a European Journal of Chemical Biology·Beyza BulutogluScott Banta
Apr 3, 2019·Advanced Healthcare Materials·Yao WangJin Kim Montclare
Mar 13, 2019·Chembiochem : a European Journal of Chemical Biology·Walaa AbdallahScott Banta
Mar 11, 2020·International Journal of Molecular Sciences·Xiuzhen GaoMingfei Liu
Oct 14, 2016·Chembiochem : a European Journal of Chemical Biology·Andy BeierUwe T Bornscheuer
Dec 21, 2017·Chemical Communications : Chem Comm·Louis LancasterIan Wheeldon
May 26, 2018·Chemical Society Reviews·Louis LancasterIan Wheeldon
Aug 17, 2017·Langmuir : the ACS Journal of Surfaces and Colloids·Xuye LangIan Wheeldon

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