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Engineering, Predicting, and Understanding Nicotinamide Cofactor Specificity


Cahn, Jackson Kenai Blender (2016) Engineering, Predicting, and Understanding Nicotinamide Cofactor Specificity. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9QV3JJ8.


Oxidoreductases, the enzymes that catalyze the transfer of electrons between molecules, represent the largest group of enzymes in metabolism, and the vast majority of these enzymes use the functionally-equivalent cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) for the storage and transport of the electrons. Understanding the interactions of these proteins with their cofactors is therefore crucial to the engineering of biological pathways and systems that involve these enzymes. In particular, because cells tightly regulate the levels of oxidized and reduced NAD and NADP, it is often valuable to engineer the specificity of enzymes to better integrate them into particular metabolic contexts.

The first section of this thesis focuses on this specificity, taking as a model system the ketol-acid reductoisomerase (KARI) enzyme family. Prior to the work described here, all known members of the KARI enzyme family displayed a strict specificity for NADP over NAD. However, the use of these enzymes in a constructed pathway for the production of medium-chain alcohols created a clear need for NAD-specific KARIs to improve yields. Chapter 1 briefly summarizes the prior state of the art in nicotinamide cofactor specificity engineering before describing how a previous switching of the KARI from E. coli was extended to create a simple recipe for the specificity reversal of any KARI enzyme. Chapter 2 then uses the insights into cofactor specificity in KARIs afforded by the engineering in Chapter 1 to search databases of KARI sequences and predict naturally NAD-specific KARIs, resulting in the discovery of extremophilic NAD-utilizing KARIs with properties that outstrip those of the best engineered enzymes. Chapter 3 extends this prediction approach into another enzyme family, xylose reductases, and discusses its strengths and limitations across diverse enzyme folds and families.

The next section of the thesis diverges from the topic of cofactor specificity engineering to briefly explore three structural questions which arise from the study of KARIs. Chapter 4 covers an analysis of several new KARI crystal structures, including those obtained during the efforts described in Chapters 1 and 2. From a comprehensive comparison of these structures, two topics are addressed: (1) the effect of insertions and deletions in the cofactor specificity loop on the binding geometry of NAD and NADP, and (2) the conformational motions involved in the binding of cofactor, substrate, and metal ions. Based on a pair of structures from Chapter 4, Chapter 5 experimentally explores the structural evolution of the KARI enzyme family’s two distinct structural classes, replicating in a class I KARI the structural duplication that produced the class II KARI fold, and demonstrating a remarkable retention of enzymatic activity. Chapter 6 explores a curious sensitivity to mutations observed around the adenine moiety of several KARIs, and extends this observation to a range of other NADP- and NAD-dependent enzymes, with implications both for engineering these proteins and for understanding protein evolution more generally.

The third and final section discusses the development of a general method for the cofactor specificity reversal of any NAD(P)-utilizing enzyme. Chapter 7 explains the approach used and how it represents a new paradigm in protein engineering, as well as covering the thorough experimental validation to which the method was subjected. This method was developed into a web applet (CSR-SALAD) for public use, and Chapter 8 discusses the development and use of this tool. This third section represents the culmination of the preceeding work, drawing from an understanding of the sequence and structural determinants of cofactor binding as explored in the preceeding six chapters to create the heuristic picture of specificity which informed the comprehensive engineering approach.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Protein engineering, biocatalysis, cofactor, nicotinamide, specificity, semi-rational engineering, structural biology, molecular evolution
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Awards:The Herbert Newby Mccoy Award, 2016
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Arnold, Frances H. (advisor)
  • Mayo, Stephen L. (co-advisor)
Group:Resnick Sustainability Institute
Thesis Committee:
  • Tirrell, David A. (chair)
  • Shan, Shu-ou
  • Dougherty, Dennis A.
  • Arnold, Frances Hamilton
  • Mayo, Stephen L.
Defense Date:19 January 2016
Non-Caltech Author (AT)
Record Number:CaltechTHESIS:01212016-225223309
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for ch. 1 adapted for ch. 2 adapted for ch. 4 adapted for ch. 5 adapted for ch. 6 ItemCh. 8. Web tool created as part of dissertation project
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9538
Deposited By: Jackson Cahn
Deposited On:08 Mar 2017 00:21
Last Modified:08 Nov 2023 00:11

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