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Mutations in adenine-binding pockets enhance catalytic properties of NAD(P)H-dependent enzymes

Cahn, J. K. B. and Baumschlager, A. and Brinkmann-Chen, S. and Arnold, F. H. (2016) Mutations in adenine-binding pockets enhance catalytic properties of NAD(P)H-dependent enzymes. Protein Engineering Design and Selection, 29 (1). pp. 31-38. ISSN 1741-0126. PMCID PMC4678007. http://resolver.caltech.edu/CaltechAUTHORS:20151030-152836183

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Abstract

NAD(P)H-dependent enzymes are ubiquitous in metabolism and cellular processes and are also of great interest for pharmaceutical and industrial applications. Here, we present a structure-guided enzyme engineering strategy for improving catalytic properties of NAD(P)H-dependent enzymes toward native or native-like reactions using mutations to the enzyme's adenine-binding pocket, distal to the site of catalysis. Screening single-site saturation mutagenesis libraries identified mutations that increased catalytic efficiency up to 10-fold in 7 out of 10 enzymes. The enzymes improved in this study represent three different cofactor-binding folds (Rossmann, DHQS-like, and FAD/NAD binding) and utilize both NADH and NADPH. Structural and biochemical analyses show that the improved activities are accompanied by minimal changes in other properties (cooperativity, thermostability, pH optimum, uncoupling), and initial tests on two enzymes (ScADH6 and EcFucO) show improved functionality in Escherichia coli.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1093/protein/gzv057DOIArticle
http://peds.oxfordjournals.org/content/29/1/31.longPublisherArticle
http://peds.oxfordjournals.org/content/29/1/31/suppl/DC1PublisherSupplementary Data
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4678007/PubMed CentralArticle
ORCID:
AuthorORCID
Arnold, F. H.0000-0002-4027-364X
Additional Information:© The Author 2015. Published by Oxford University Press. Received September 25, 2015. Accepted September 28, 2015. First published online: October 27, 2015. The authors thank Nelson Chou and Drs Sheel Dodani, Tillmann Heinisch and Devin Trudeau for their experimental assistance; Drs Devin Trudeau and John McIntosh for their helpful discussions; and Prof. Dan Tawfik, Prof. Pat Cirino and Prof. Monica Gerth for their helpful comments on this manuscript. They also thank Dr Jens Kaiser and Pavle Nicolovski of the Caltech Molecular Observatory for their continued support. This work was supported by the Gordon and Betty Moore Foundation through grant number GBMF2809 to the Caltech Programmable Molecular Technology Initiative and by American Recovery and Reinvestment Act (ARRA) funds through the National Institutes of Health Shared Instrumentation Grant Program, grant number S10RR027203, to F.H.A. J.K.B.C. acknowledges the support of the Resnick Sustainability Institute (Caltech).
Group:Resnick Sustainability Institute
Funders:
Funding AgencyGrant Number
Gordon and Betty Moore FoundationGBMF2809
American Recovery and Reinvestment Act (ARRA)UNSPECIFIED
NIHS10RR027203
Resnick Sustainability InstituteUNSPECIFIED
Subject Keywords:directed evolution, enzyme optimality, nicotinamide cofactors, protein engineering, site-saturation mutagenesis
PubMed Central ID:PMC4678007
Record Number:CaltechAUTHORS:20151030-152836183
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20151030-152836183
Official Citation:J.K.B. Cahn, A. Baumschlager, S. Brinkmann-Chen, and F.H. Arnold Mutations in adenine-binding pockets enhance catalytic properties of NAD(P)H-dependent enzymes Protein Engineering, Design and Selection (2016) 29 (1): 31-38 first published online October 27, 2015 doi:10.1093/protein/gzv057
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:61753
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:02 Nov 2015 21:34
Last Modified:18 Jul 2017 20:40

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