A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases

Abstract

Supporting Table 1. NADP-to-NAD cofactor specificity reversal. Structures followed by (h) are homology models, while those followed by another PDB accession code use the cofactor from that protein and (m) denotes a structure of a mutant protein. Mutations indicated in italics are distal to the 2' position. For citation information, see Supporting Material 2.

Switching the Cofactor Specificity of an Imine Reductase

“…Cahn et al. demonstrated in previous work the robustness of CSR‐SALAD including one protein without a crystal structure . Recommended mutations for R ‐IRED_ Sk at seven positions were transferred onto R ‐IRED_ Ms .…”

“…To address this important challenge of improving the specificity and activity for NADH, we altered the cofactor specificity of the R ‐selective IRED from Myxococcus stipitatus ( R ‐IRED_ Ms ) by using the recently described online tool “Cofactor Specificity Reversal—Structural Analysis and Library Design” (CSR‐SALAD), developed in the lab of Frances H. Arnold. CSR‐SALAD is a semi‐rational structure‐guided approach that aims at reversing the nicotinamide cofactor specificity of enzymes with Rossman fold binding pockets as in the case of IREDs (Figure ) . This approach includes the simultaneous mutagenesis of phosphate coordinating residues, which generally determine the cofactor specificity.…”

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

“…The most common fold employed by these enzymes for such purpose is the Rossmann fold, but there is a variety of other less common structural motifs which can also bind the redox cofactor, such as the TIMbarrel, the dihydroquinoate synthase-like and the FAD/NAD + binding folds [66].…”

“…There have also been multiple attempts to modify the specificity of NAD(P)H-dependent oxidoreductases for the cofactor they use for the electron transfer reaction, as controlling the cofactor specificity of this class of enzymes can be used to optimally engineer the cellular metabolism by achieving a better balance of cofactor availability [171,172]. During the last two decades, there have been a considerable number of successful cases of relaxation or even reversal of NADH/NADPH cofactor specificity.…”

“…Furthermore, the ability to exploit this increasing knowledge base has been facilitated by the development of protein engineering and directed evolution approaches which allow the properties of enzymes to be tailored in rational and semi-rational ways. There has been practical progress in the prediction of enzyme function from structure: The new CSR-SALAD computational tool uses recently-established rules to interpret structural and sequence information in order to predict specific mutations which might allow reversal of the cofactor specificity of a given NAD(P)H-dependent oxidoreductase [66]. The rapid growth in sequence and structural information together with technological developments will undoubtedly expand still further the potential and usefulness of an already naturally versatile group of enzymes.…”