Re‐engineering the discrimination between the oxidized coenzymes NAD+ and NADP+ in clostridial glutamate dehydrogenase and a thorough reappraisal of the coenzyme specificity of the wild‐type enzyme

  title={Re‐engineering the discrimination between the oxidized coenzymes NAD+ and NADP+ in clostridial glutamate dehydrogenase and a thorough reappraisal of the coenzyme specificity of the wild‐type enzyme},
  author={Marina Capone and David O. Scanlon and Joanna Griffin and Paul C. Engel},
  journal={The FEBS Journal},
Clostridial glutamate dehydrogenase mutants, designed to accommodate the 2′‐phosphate of disfavoured NADPH, showed the expected large specificity shifts with NAD(P)H. Puzzlingly, similar assays with oxidized cofactors initially revealed little improvement with NADP+, although rates with NAD+ were markedly diminished. This article reveals that the enzyme’s discrimination in favour of NAD+ and against NADP+ had been greatly underestimated and has indeed been abated by a factor of > 16 000 by the… 
Glutamate Dehydrogenases: The Why and How of Coenzyme Specificity
  • P. Engel
  • Biology
    Neurochemical Research
  • 2013
Structural studies show NAD+-dependent, NADP+- dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity.
Reversal of the extreme coenzyme selectivity of Clostridium symbiosum glutamate dehydrogenase
Three mutations of glutamate dehydrogenase, previously identified as having a potential bearing on coenzyme specificity, did not engender any further shift in preference when incorporated into the triple mutant, despite having a significant effect when expressed as a single mutant.
Structure of NADP+‐dependent glutamate dehydrogenase from Escherichia coli – reflections on the basis of coenzyme specificity in the family of glutamate dehydrogenases
These studies clarify the sequence–structure relationships in bacterial GDHs, revealing that identical residues may specify different coenzyme preferences, depending on the structural context, and primary sequence alone is therefore not a reliable guide for predicting coen enzyme specificity.
Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges
This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes.
A General Tool for Engineering the NAD/NADP Cofactor Preference of Oxidoreductases.
This work presents a structure-guided, semirational strategy for reversing enzymatic nicotinamide cofactor specificity that leverages the diversity and sensitivity of catalytically productive cofactor binding geometries to limit the problem to an experimentally tractable scale.
Structure-based Conversion of the Coenzyme Requirement of a Short-chain Dehydrogenase/Reductase Involved in Bacterial Alginate Metabolism*
It is shown that the two loops determine the coenzyme requirement, and loop exchange is a promising method for conversion of coen enzymatically characterized enzymes in the SDR family.


Complete Reversal of Coenzyme Specificity of Xylitol Dehydrogenase and Increase of Thermostability by the Introduction of Structural Zinc*
The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability and further increased the catalytic activity with NADP+.
Redesign of the coenzyme specificity in L-lactate dehydrogenase from bacillus stearothermophilus using site-directed mutagenesis and media engineering.
The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild- type LDH in a reaction mixture containing 15% methanol, and the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH.
An Examination by Site-Directed Mutagenesis of Putative Key Residues in the Determination of Coenzyme Specificity in Clostridial NAD+-Dependent Glutamate Dehydrogenase
Sequence and structure comparisons of various glutamate dehydrogenases (GDH) and other nicotinamide nucleotide-dependent dehydrogenases have potentially implicated certain residues in coenzyme
Change of nucleotide specificity and enhancement of catalytic efficiency in single point mutants of Vibrio harveyi aldehyde dehydrogenase.
The results have shown that the size and the structure of the residue at the nucleotide recognition site play the key roles in differentiating between NAD(+) and NADP(+) interactions while the presence of a negative charge is responsible for the decrease in interactions with NADp(+) in Vh-ALDH.
A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity.
  • R. Chen, A. Greer, A. Dean
  • Biology
    Proceedings of the National Academy of Sciences of the United States of America
  • 1995
The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD, so site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD.
Alteration of the specificity of the cofactor-binding pocket of Corynebacterium 2,5-diketo-D-gluconic acid reductase A.
To the best of the authors' knowledge, this is the first focused attempt to alter the cofactor specificity of a member of the aldo-keto reductase superfamily by engineering improved activity with NADH into the enzyme.
Relaxing the nicotinamide cofactor specificity of phosphite dehydrogenase by rational design.
The combined results provide clear evidence that Glu175 and Ala176 are both critical for nicotinamide cofactor specificity, and the rationally designed double mutant might be useful for the development of an efficient in vitro NAD(P)H regeneration system for reductive biocatalysis.
The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography.
Site-directed mutagenesis is used to replace interactions specific to the enzyme-NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity.