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A general Tool for

Engineering the NAD/NADP

Cofactor Preference of
Oxidoreductases *
Jackson K. B. Cahn, Caroline A. Werlang, Armin Baumschlager, Sabine Brinkmann-Chen,
Stephen L. Mayo, and Frances H. Arnold
California Institute of Technology

Daniel Alejandro Caballero Cerbon

* Or how to engineer enzymes with a salad.


The model
Experimental validation
Paper conclusions
Personal conclusions

Spatially distant from the

nicotinamid group.
Plays no role in the enzymatic
However there is preference (even
inside an enzyme family) ->
Increase pathway yield.
Minimization of side products.
Improving equilibrium metabolite levels.

Marginal success
Sensitivity to Amino acid exchange Activity reduction
to cofactor exchange
Structural diversity of cofactor binding-and specificity musters. (even between
enzymes of the same family)
Random mutagenesis and Screening, multiple simultaneous mutations required (too big
libraries) + non-additivity of the mutations effects.
Cofactor specificity inside
an enzyme family
Ketol-acid reductoisomerase (KARI) family.
NADP preferent
Exchange to NAD in evolutionary history.
Every cofactor exchange was carried out through an one-of-a-kind combination of
amino acid exchanges, insertions and deletions.
Trend between cofactor preference and charge or polarity of the binding site.
NADP: Positively charged amino acids (Arg, Leu), H-bond donors
NAD: Negatively charged amino acids (Asp, Glu)
Cofactor Specificity Reversal Structural
Analysis and Library Design
Detect residues that affect cofactor specificity.
Classify based on position and orientation
Design small degenerate codon library
User control of library size
Identify structural hotspots for compensatory mutations, activity recovery.

Structural biology, biophysics and heuristics.

Homology based models using SWISS-MODEL server.

Specificity determining residues:

Direct contact with the C2 moiety at the adenine end.
Contact through water mediated interactions.
In the case of NADP, contact with the phosphat in C2.

Classification by their role in the binding site formation.

Residues that interact frontally with the adenine ring.
Residues that interact sideways with tthe adenine ring.
Residues thet interact with both the C2 moiety and the C3 OH- group.
Library of mutants in the specifity determining positions.
Subsaturation degenerate codons libraries.
Every residue in every class posess a range of degenerate codons, which code
for a spceific number of amino acids -> User determinde library size.
Mutations to structurally similar reidues, which were empirically identified as
useful for the specificity exchange.


Objective: Enzyme activity recovery.
Compensatory mutations. Restabilization and
Structural prediction of positions for compensatory
mutations. Site directed mutagenesis.
Mutations around the adenine ring.

High priority: left out due to library size.

Medium priority: Around adenine but not for specificity
Low priority: Hidrogen bonds with specificity residues
Experimental Validation
4 Oxidoreduktases: Glyoxylat Reductase (GR), Cinnamyl Alcohol Dehydrogenase
(CinADH), Xylose Reductase (XR) and Iron containing Alcohol Dehydrogenase (FeADH).

NADP preferring Enzymes. Exchange to NAD more complicated and relevant for
industrial applications

Creation of the mutant library with the codons recomended by CSR-SALAD for the
specificity Exchange. Escherichia coli BL21

0-2 rounds of Saturation mutagenesis for the best mutants for the activity recovery.
Library creation
1. Quikchange
2. Single colonies picking + (300 l Luria broth 100g/ml Ampicilin) -> 96 well
Overnight, 37C, 225 rpm, 80% humidity.
3. 50l preculture + 600l LB-Amp
3h, 37C
4. + 50l LB-Amp + IPTG -> Expression
5. Bradford Assay ->Protein concentration

6. Activity measurements with NADH absorbance 340 nm


Activity Recovery
= 20%

= 630%

= 3%

= 316%
Results: Previous studies recapitulation

Comparison with literature.

CSR-SALAD reliably produces the desired enzyme properties with minimum
expertise and labor.
Multiple mutational solutions for each specificity reversal problem.
CSR-SALAD will be useful in the fields of synthetic biology metabolic engineering
and biocatalists.
Will make cofactor reversal a routine task rather than a formidable engineering
Ingenious way to combine minimal theory bases with an heuristic model.
Algorithm: user defined library size to fit experimental limitations.
Should have also performed validation with switch to NADP.
Make validation for specificity and activity separately.
Significance of the recapitulation? How effective where the performed mutations?
How well can this method be applied to other cofactors?