University of Oulu

Sridhar, S., Zavarise, A., Kiema, T.-R., Dalwani, S., Eriksson, T., Hajee, Y., Reddy Enugala, T., Wierenga, R. K., & Widersten, M. (2023). Crystal structures and kinetic studies of a laboratory evolved aldehyde reductase explain the dramatic shift of its new substrate specificity. IUCrJ, 10(4), 437–447.

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

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Author: Sridhar, Shruthi1,2,3; Zavarise, Alberto1; Kiema, Tiila-Riikka3;
Organizations: 1Department of Chemistry – BMC, Uppsala University, SE-751 23 Uppsala, Sweden
2Faculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5400, Oulu FI-90014, Finland
3Biocenter Oulu, University of Oulu, PO Box 5000, Oulu FI-90014, Finland
Format: article
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 1.1 MB)
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Language: English
Published: International Union of Crystallography, 2023
Publish Date: 2023-09-19


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.

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Series: IUCrJ
ISSN: 2052-2525
ISSN-E: 2052-2525
ISSN-L: 2052-2525
Volume: 10
Issue: Pt 4
Pages: 437 - 447
DOI: 10.1107/S205225252300444X
Type of Publication: A1 Journal article – refereed
Field of Science: 116 Chemical sciences
Funding: This work was supported by grants of the Olle Engkvist Byggma ̈ stare Foundation (grant no. 194–0638 to MW) and of the Academy of Finland (grant no. 339894 to RKW).
Academy of Finland Grant Number: 339894
Detailed Information: 339894 (Academy of Finland Funding decision)
Copyright information: © The Author(s) 2023. Published under a CC BY 4.0 licence.