Molecular Dynamics Analysis of a Rationally Designed Aldehyde Dehydrogenase Gives Insights into Improved Activity for the Non-Native Cofactor NAD⁺

Abstract

The aldehyde dehydrogenase from Thermoplasma acidophilum was previously implemented as a key enzyme in a synthetic cellfree 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.

Short-Chain Dehydrogenase NcmD Is Responsible for the C-10 Oxidation of Nocamycin F in Nocamycin Biosynthesis

“…The current results confirm that Asp37-containing NcmD prefers NAD + (Kallberg et al, 2002), which has been confirmed by resultant in vitro enzymatic assays. Generally, the catalytic activities decrease significantly when the SDRs are not compatible with cofactors, which have been observed from NcmD enzymatic assays, and similar results are also revealed from other SDRs (Moon et al, 2012;Takase et al, 2014;Cao et al, 2019;Gmelch et al, 2020).…”

Integrating Carbohydrate and C1 Utilization for Chemicals Production

“…Transketolase (TktA) kinetics (Figure S2) were measured in a coupled approach with glyceraldehyde specific aldehyde dehydrogenase taAldH (M42) [50] by measuring reduction of NAD + . To test whether M42 activity is reduced by formaldehyde, activity of M42 was tested on glyceraldehyde by measuring the reduction of NAD + in presence of 0 to 20 mM formaldehyde (Figure S3).…”

Stairway to Stereoisomers: Engineering Short‐ and Medium‐Chain Ketoreductases To Produce Chiral Alcohols

“…Gluconobacter oxydans 621H SDR low promiscuity WP_172493137 [143] PyAeADHII Pyrobaculum aerophilum MDR low promiscuity 4JBH [30] PasCR Pichia pastoris GS115 SDR promiscuous XP_002491455 [144] TaAIDH Thermoplasma acidophilum ALDH_SF multiple substrates CAC11938 [145][146][147] AtADH Arabidopsis thaliana MDR multiple substrates P06525 [148] BgADH3 Burkholderia gladioli CCTCC M2012379 SDR low promiscuity WP_172493137 [149] AcCR Acetobacter sp. CCTCC M209061 SDR multiple substrates MF419650 [150] ChKRED20 Chryseobacterium sp.…”