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

Area of Science:

• Biochemistry
• Bioinformatics
• Enzymology

Background:

• Enzyme discovery is often difficult, hindering biocatalysis applications.
• Understanding enzyme sequence-function relationships is crucial for protein engineering.

Purpose of the Study:

• To present a computational approach (A²CA) integrating phylogenetic information and multiple sequence alignments (MSA) for novel enzyme discovery.
• To identify sequence-function relationships within the 4-phenol oxidase enzyme family.

Main Methods:

• Utilized the A²CA tool to connect phylogenetic data and MSA for enzyme discovery.
• Focused on the VAO/PCMH flavoprotein family, specifically 4-phenol oxidases, using known crystal structures.
• Analyzed active site residues and performed site-saturation mutagenesis on selected bacterial 4-phenol oxidases.

Main Results:

• Characterization of eight bacterial 4-phenol oxidases revealed substrate scope differences linked to key active site residues.
• Site-saturation mutagenesis yielded mutants with enhanced activity on specific substrates.
• Mutants were generated that could accept non-natural substrates, demonstrating altered enzyme function.

Conclusions:

• The A²CA approach effectively aids in discovering new enzymes and elucidating sequence-function links.
• Active site residue modifications can tune substrate specificity and introduce novel substrate acceptance in enzymes.
• This method facilitates the engineering of biocatalysts for specific applications.