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Introduction
The model
Experimental validation
Results
Conclusions
Paper conclusions
Personal conclusions
NAD vs. NADP
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
Strategy
Analyse
Detect residues that affect cofactor specificity.
Classify based on position and orientation
Switch
Design small degenerate codon library
User control of library size
Recover
Identify structural hotspots for compensatory mutations, activity recovery.
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
plates
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
Activity Recovery
= 20%
= 630%
= 3%
= 316%
Results: Previous studies recapitulation