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Target Concepts:
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Query: EC:6.2.1.1 (
ACS
)
78,556
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
The aldehyde dehydrogenase from
Thermoplasma acidophilum
was previously implemented as a key enzyme in a synthetic cell-free reaction cascade for the production of alcohols. In order to engineer the enzyme's cofactor specificity from NADP
+
to NAD
+
, we identified selectivity-determining residues with the
CSR
-SALAD tool and investigated further positions based on the crystal structure. Stepwise combination of the initially discovered six point mutations allowed us to monitor the cross effects of each mutation, resulting in a final variant with reduced
K
M
for the non-native cofactor NAD
+
(from 18 to 0.6 mM) and an increased activity for the desired substrate d-glyceraldehyde (from 0.4 to 1.5 U/mg). Saturation mutagenesis of the residues at the entrance of the substrate pocket could eliminate substrate inhibition. Molecular dynamics simulations showed a significant gain of flexibility at the cofactor binding site for the final variant. The concomitant increase in stability against isobutanol and only a minor reduction in its temperature stability render the final variant a promising candidate for future optimization of our synthetic cell-free enzymatic cascade.
ACS
Synth Biol 2020 04 17
PMID:Molecular Dynamics Analysis of a Rationally Designed Aldehyde Dehydrogenase Gives Insights into Improved Activity for the Non-Native Cofactor NAD
.
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