Substrate specificity of 2-deoxy-D-ribose 5-phosphate aldolase (DERA) assessed by different protein engineering and machine learning methods

Abstract

In this work, deoxyribose-5-phosphate aldolase (Ec DERA, EC 4.1.2.4) from Escherichia coli was chosen as the protein engineering target for improving the substrate preference towards smaller, non-phosphorylated aldehyde donor substrates, in particular towards acetaldehyde. The initial broad set of mutations was directed to 24 amino acid positions in the active site or in the close vicinity, based on the 3D complex structure of the E. coli DERA wild-type aldolase. The specific activity of the DERA variants containing one to three amino acid mutations was characterised using three different substrates. A novel machine learning (ML) model utilising Gaussian processes and feature learning was applied for the 3rd mutagenesis round to predict new beneficial mutant combinations. This led to the most clear-cut (two- to threefold) improvement in acetaldehyde (C2) addition capability with the concomitant abolishment of the activity towards the natural donor molecule glyceraldehyde-3-phosphate (C3P) as well as the non-phosphorylated equivalent (C3). The Ec DERA variants were also tested on aldol reaction utilising formaldehyde (C1) as the donor. Ec DERA wild-type was shown to be able to carry out this reaction, and furthermore, some of the improved variants on acetaldehyde addition reaction turned out to have also improved activity on formaldehyde. Key points • DERA aldolases are promiscuous enzymes. • Synthetic utility of DERA aldolase was improved by protein engineering approaches. • Machine learning methods aid the protein engineering of DERA.

Current state of and need for enzyme engineering of 2-deoxy-D-ribose 5-phosphate aldolases and its impact

“…through Ser239Asp mutation, also likely contributed to the decreased phosphate binding. All the best DERA variants included also mutation at Cys47, which has been shown also in other studies to be a beneficial target for substitutions, probably due to decreased covalent inhibitory adduct formation with aldehydes (Voutilainen et al 2020 ). The DERA from a hyperthermophilic A. pernix has naturally a Valine residue at this position.…”

“…In general, the thermostabilities of DERA enzymes seem to vary for reasons that are not self-evident. As an example, DERA derived from E. coli , a mesophilic bacterium, is relatively thermostable, having the measured melting temperature ( T m ) of 65 °C (Voutilainen et al 2020 ).…”

“…Voutilainen et al have recently used three rounds of mutagenesis combined with a ML method to create variants of E. coli DERA having improved catalytic activity for acetaldehyde aldol reaction, combined with clearly reduced activity for DR5P and DR retroaldol reaction (Voutilainen et al 2020 ). At first round, 69 single DERA mutants were created targeting 24 amino acid positions selected mainly by using 3D structures, literature data, and bioinformatics tools, such as Hotspot Wizard (Bendl et al 2016 ).…”

“…The results from the first and second round contained biochemical characterization and sequence data for 131 E. coli DERA variants, which was used to train the ML model to automatically predict all three substrate specificities (on DR5P, DR, and acetaldehyde) (Voutilainen et al 2020 ). Altogether 48,000 new E. coli DERA variants having 1–3 amino acid mutations were then created and screened in silico.…”

“…The key catalytic residue is Lysine which make a covalent bond with the donor aldehyde. In addition, there are other residues in the active site which promote the reaction participating in protonation and deprotonation of intermediates measured melting temperature (T m ) of 65°C (Voutilainen et al 2020).…”