Substrate scope and selectivity in offspring to an enzyme subjected to directed evolution

Abstract

We have analyzed the effects of mutations inserted during directed evolution of a specialized enzyme, Escherichia coli S-1,2-propanediol oxidoreductase (FucO). The kinetic properties of evolved variants have been determined and the observed differences have been rationalized by modeling the tertiary structures of isolated variants and the wild-type enzyme. The native substrate, S-1,2-propanediol, as well as phenylacetaldehyde and 2S-3-phenylpropane-1,2-diol, which are new substrates accepted by isolated variants, were docked into the active sites. The study provides a comprehensive picture of how acquired catalytic properties have arisen via an intermediate generalist enzyme, which had acquired a single mutation (L259V) in the active site. Further mutagenesis of this generalist resulted in a new specialist catalyst. We have also been able to relate the native enzyme activities to the evolved ones and linked the differences to individual amino acid residues important for activity and selectivity. F254 plays a dual role in the enzyme function. First, mutation of F254 into an isoleucine weakens the interactions with the coenzyme thereby increasing its dissociation rate from the active site and resulting in a four-fold increase in turnover number with S-1,2-propanediol. Second, F254 is directly involved in binding of aryl-substituted substrates via p-p interactions. On the other hand, N151 is critical in determining the substrate scope since the side chain amide group stabilizes binding of 1,2-substituted diols and is apparently necessary for enzymatic activity with these substrates. Moreover, the side chain of N151 introduces steric hindrance, which prevents high activity with phenylacetaldehyde. Additionally, the hydroxyl group of T149 is required to maintain the catalytically important hydrogen bonding network.

Relaxation of nonproductive binding and increased rate of coenzyme release in an alcohol dehydrogenase increases turnover with a nonpreferred alcohol enantiomer

“…We have observed similar behavior, that is, apparent rate increase in laboratory‐evolved enzyme variants coupled with higher turnover values with a parallel raise in K M values, also in other alcohol dehydrogenases and hydrolases . Nonproductive binding (of a nonpreferred substrate isomer) may be a general phenomenon in substrate discrimination and promiscuity , with enantioselectivity as an extreme example.…”

“…The resuspended samples were centrifuged at 12 270 g in a microcentrifuge for 5 min and subsequently transferred to HPLC glass vials together with reference samples. The amounts of ( R )‐ and ( S )‐ 1 were then analyzed by chiral HPLC (Chiralpack AS‐H, 250 × 4.6 mm Ø) using a n‐hexane/isopropanol (90 : 10 v/v) mixture as the mobile phase, a method adapted from . The system used an LC 20AD solvent delivery module (Shimadzu, Tokyo, Japan), a Shimadzu SIL‐10AF autosampler, and a Shimadzu SPDM20A photometric unit with a 0.5 mL·min −1 flow rate and 20‐min analysis time for each sample.…”

A Microplate Format Assay for Real‐Time Screening for New Aldolases that Accept Aryl‐Substituted Acceptor Substrates

“…We have, in ap arallel directed evolution effort, isolated an alcohol dehydrogenasev ariant of E. coli propane-1,2-diol oxidore-ductaseF ucO, dubbed DA1472. [13] DA1472 contains two point mutations (N151G, L259V) that renderi tv irtually inactive with (2S)-propane-1,2-diol, the natural substrate of FucO, [14] but highly active with 2.F urthermore, DA1472 does not accept secondary alcohols or ketones as substrates andt herefore does not act on aldol 1 either in oxidation or in reduction reactions. Hence, DA1472 exhibits all properties necessary for inclusion as ar eporter( coupling) enzyme for FSA-catalyzed cleavage of 1.…”

Molecular characterization of central cytoplasmic loop in Aspergillus nidulans AstA transporter

“…The mutations introduced in substrate binding site of MFS transporters usually lead to a pronounced 10-fold K m alteration (Will et al, 1998;Sahin-Tóth et al, 1999). Among some previously described enzymes, S-1,2-propanediol oxidoreductase (FucO) alters its substrate specificity substantially upon just a single point mutation L 259 V in the substrate-binding site, while its K m increases just two-fold (Blikstad et al, 2014). In case of the higher eukaryotes, some mutated enzymes, it was shown that human galactokinase, point-mutated in its substrate-binding site (A 198 V), has K m only weakly altered (30% decrease) and its kinetic parameters are almost comparable to the wild type (Timson & Reece, 2003).…”