Hydroxamate Assays for High‐Throughput Screening of Transketolase Libraries Against Arylated Substrates

We recently reported that the transketolase from Geobacillus stearothermophilus (TKgst) upon acyl transfer to nitrosoarenes generates N‐aryl hydroxamic acids (HA). The latter are metal chelating compounds that in the presence of Fe(III) ions form deep‐red complexes. Here, we applied this principle t...

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Bibliographic Details
Published in:Advanced synthesis & catalysis 2023-11, Vol.365 (22), p.3861-3871
Main Authors: Fúster Fernández, Inés, Kickstein, Michael, Fessner, Wolf‐Dieter
Format: Article
Language:English
Subjects:
Aldehydes
Assaying
Chelation
colorimetric assay
high-throughput screening
hydroxamic acids
Iron
iron (III) chelation
Screening
Solid surfaces
Substrates
transketolase
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Summary:We recently reported that the transketolase from Geobacillus stearothermophilus (TKgst) upon acyl transfer to nitrosoarenes generates N‐aryl hydroxamic acids (HA). The latter are metal chelating compounds that in the presence of Fe(III) ions form deep‐red complexes. Here, we applied this principle to the development of a colorimetric assay in both solid‐ and liquid‐phase formats for the high‐throughput screening of TKgst and its variants. Screening a set of positive hits from a L382X/D470X library validated the specificity and sensitivity of the assays. The solid surface assay allows a clear distinction between positive and negative colonies by the naked eye in qualitative mode, and further also to measure activity in semi‐quantitative fashion in the liquid‐phase format. The assay will be important for engineering the TKgst enzyme towards improved conversion of aromatic aldehydes by their close structural analogy to nitrosoarenes.
ISSN:1615-4150
1615-4169
DOI:10.1002/adsc.202300989