Article Metrics


Online attention

Crystal structures and kinetic studies of a laboratory evolved aldehyde reductase explain the dramatic shift of its new substrate specificity

DOI: 10.1107/S205225252300444X DOI Help

Authors: Shruthi Sridhar (Uppsala University; University of Oulu) , Alberto Zavarise (Uppsala University) , Tiila-Riikka Kiema (University of Oulu) , Subhadra Dalwani (University of Oulu) , Tor Eriksson (Uppsala University) , Yannick Hajee (Uppsala University) , Thilak Reddy Enugala (Uppsala University) , Rik. K. Wierenga (University of Oulu) , Mikael Widersten (Uppsala University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Iucrj , VOL 10

State: Published (Approved)
Published: July 2023

Open Access Open Access

Abstract: The Fe2+-dependent E. coli enzyme FucO catalyzes the reversible interconversion of short-chain (S)-lactaldehyde and (S)-1,2-propane­diol, using NADH and NAD+ as cofactors, respectively. Laboratory-directed evolution experiments have been carried out previously using phenyl­acetaldehyde as the substrate for screening catalytic activity with bulky substrates, which are very poorly reduced by wild-type FucO. These experiments identified the N151G/L259V double mutant (dubbed DA1472) as the most active variant with this substrate via a two-step evolutionary pathway, in which each step consisted of one point mutation. Here the crystal structures of DA1472 and its parent D93 (L259V) are reported, showing that these amino acid substitutions provide more space in the active site, though they do not cause changes in the main-chain conformation. The catalytic activity of DA1472 with the physiological substrate (S)-lactaldehyde and a series of substituted phenyl­acetaldehyde derivatives were systematically quantified and compared with that of wild-type as well as with the corresponding point-mutation variants (N151G and L259V). There is a 9000-fold increase in activity, when expressed as kcat/KM values, for DA1472 compared with wild-type FucO for the phenyl­acetaldehyde substrate. The crystal structure of DA1472 complexed with a non-reactive analog of this substrate (3,4-di­meth­oxy­phenyl­acetamide) suggests the mode of binding of the bulky group of the new substrate. These combined structure–function studies therefore explain the dramatic increase in catalytic activity of the DA1472 variant for bulky aldehyde substrates. The structure comparisons also suggest why the active site in which Fe2+ is replaced by Zn2+ is not able to support catalysis.

Journal Keywords: aldehyde reductase; enzyme functions; enzyme mechanisms; directed evolution; substrate selectivity; FucO

Diamond Keywords: Enzymes; Bacteria

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I24-Microfocus Macromolecular Crystallography

Other Facilities: BioMAX at MAX IV

Added On: 07/06/2023 09:30


Discipline Tags:

Evolutionary science Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)