Sequence-function relation for the prediction of enzyme properties: A case study on flavin-dependent oxidases

The discovery of enzymes (biocatalysts) can sometimes be challenging. Herein, we describe an approach for connecting phylogenetic information and multiple sequence alignments (MSA), via the use of a small computational tool called A²CA, for the discovery of new enzymes and how it can be used to find sequence-function relationships within a protein family. We chose 4-phenol oxidases of the VAO/PCMH flavoprotein family to demonstrate this approach, as there were already three crystal structures of this enzyme family known, which allows for an easier narrowing of residues to look at during the process. Focusing on first shell amino acids of the active site, we selected eight bacterial members of the 4-phenol oxidases. Characterizations of their substrate scopes revealed clear differences that could be linked to in key residues in the active sites. Information gained on key residues were used to perform site-saturation mutagenesis, resulting in mutants that either showed improved activities towards specific substrates or even allowed for the acceptance of non-natural substrates.