Repurposing designed mutants: a valuable strategy for computer-aided laccase engineering – the case of POXA1b

Abstract

The Q3broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates.Please check this proof carefully. Our staff will not read it in detail after you have returned it.Translation errors between word-processor files and typesetting systems can occur so the whole proof needs to be read. Please pay particular attention to: tabulated material; equations; numerical data; figures and graphics; and references. If you have not already indicated the corresponding author(s) please mark their name(s) with an asterisk. Please e-mail a list of corrections or the PDF with electronic notes attached -do not change the text within the PDF file or send a revised manuscript. Corrections at this stage should be minor and not involve extensive changes. All corrections must be sent at the same time.Please bear in mind that minor layout improvements, e.g. in line breaking, table widths and graphic placement, are routinely applied to the final version.Please note that, in the typefaces we use, an italic vee looks like this: n, and a Greek nu looks like this: ν.We will publish articles on the web as soon as possible after receiving your corrections; no late corrections will be made.Please return your final corrections, where possible within 48 hours of receipt, by e-mail to: catalysis@rsc.org Queries for the attention of the authors The broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates. In this work, a computational methodology that accumulates beneficial interactions between the enzyme and the substrate in productive conformations is applied to oxidize 2,4-diamino-benzenesulfonic acid with POXA1b laccase. Although the experimental validation of two designed variants yielded negative results, most likely due to the hard oxidizability of the target substrate, molecular simulations suggest that a novel polar binding scaffold was designed to anchor negatively charged groups. Consequently, the oxidation of three such molecules, selected as representative of different classes of substances with different industrial applications, significantly improved. According to molecular simulations, the reason behind such an improvement lies in the more productive enzyme-substrate binding achieved thanks to the designed polar scaffold. In the future, mutant repurposing toward other substrates could be first carried out computationally, as done here, testing molecules that share some similarity with the initial target. In this way, repurposing would not be a mere safety net (as it is in the laboratory and as it was here) but rather a powerful approach to transform laccases into more efficient multitasking enzymes.

A structural-chemical explanation of fungal laccase activity

“…In another study, K m values were reported for the activity of CuL toward phenols, acids, ketones, amines and phosphates 29 . Structure-guided computational design of laccases using both classical and quantum mechanical approaches is a viable and very useful path to discover new proficient laccases for turnover of specific substrates 30 , 31 . Quantitative models trained on actual turnover data would in principle offer more predictive accuracy, but depend critically on systematic experimental data on substrate turnover rates for their validation.…”

Green synthesis of conductive polyaniline by Trametes versicolor laccase using a DNA template

“…Despite a disadvantageous ΔE° between enzyme and substrate, all the tested laccases proved to be able to oxidize aniline, although with different efficiency (Figure ). This result further highlights how laccase reactivity is an intricate multidimensional problem in which the Electron Transfer driving force is an important factor depending on not only T1 E°, but also on the right pose of the substrate within the binding pocket. Particularly relevant is the difference observed between rPOXA1b and 1H6C, where the latter enzyme, although characterized by a higher redox potential with respect to the wild type, displayed the worst performance in aniline oxidation.…”

Directed Evolution of a Homodimeric Laccase from Cerrena unicolor BBP6 by Random Mutagenesis and In Vivo Assembly

“…The above results suggest that the performance of the mutants is not proportional to the number of substitutions. Interestingly, the kinetics performance of the laccase variants towards ABTS was considerably enhanced, while their affinity constants were similar to those of other fungal laccases [ 17 , 30 ].…”