Abstract
Discovery of novel enzymes is a challenging task, yet a crucial one, due to their increasing relevance as chemical catalysts and biotechnological tools. In our work we present a high‐throughput screening approach to discovering novel activities. A screen of 96 putative oxidases with 23 substrates led to the discovery of two new enzymes. The first enzyme, N‐acetyl‐D‐hexosamine oxidase (EC 1.1.3.29) from Ralstonia solanacearum, is a vanillyl alcohol oxidase‐like flavoprotein displaying the highest activity with N‐acetylglucosamine and N‐acetylgalactosamine. Before our discovery of the enzyme, its activity was an orphan one ‐ experimentally characterized but lacking the link to amino acid sequence. The second enzyme, from an uncultured marine euryarchaeota, is a long‐chain alcohol oxidase (LCAO, EC 1.1.3.20) active with a range of fatty alcohols, with 1‐dodecanol being the preferred substrate. The enzyme displays no sequence similarity to previously characterised LCAOs, and thus is a completely novel representative of a protein with such activity.
Highlights
Enzymes are macromolecules that catalyse chemical reactions in living organisms
We explored the activity profile of sequences annotated as 1.1.3.X oxidoreductases by using a large-scope, high-throughput screening approach
In this work we discovered two novel enzymes: one orphan enzyme, for which only the activity, but not the sequence was known, and one non-homologous isofunctional enzyme, performing a known activity, but being evolutionarily unrelated to previously described representatives
Summary
Years of enzyme studies have provided great insight into their significance in metabolism and disease.[1] Many enzymes are indispensable in food, agriculture, chemical and pharmaceutical industries.[2,3] Finding novel enzymatic activities, remains a challenging task. Nontargeted in vitro metabolomics is an example of an approach that enables screening for a wide range of activities. This method resulted in annotation of many enzymes with unknown functions, labelling the majority of metabolites without ambiguity proves difficult.[9,10] Each approach to finding new enzymatic activities has its advantages and disadvantages; together with the ever-evolving bioinformatic tools, they complement each other in this challenging task.[11,12,13]
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