Structural and functional analysis of l-methionine oxidase identified through sequence data mining.

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• l-Amino acid oxidases (LAAOs) are FAD-dependent enzymes catalyzing l-amino acid oxidation.
• Existing LAAO characterization primarily focuses on enzymes specific for charged or aromatic l-amino acids.
• LAAOs with high specificity for alkyl-chain l-amino acids, like l-methionine (l-Met), remain poorly understood.

Purpose of the Study:

• To screen and identify novel LAAOs with high specificity for l-Met.
• To characterize the biochemical and structural properties of these l-Met-specific LAAOs.
• To elucidate the molecular basis of substrate specificity in LAAOs.

Main Methods:

• Sequence similarity network (SSN) analysis for screening bacterial LAAOs.
• Enzyme activity assays to determine substrate specificity and optimal conditions.
• X-ray crystallography to determine the 3D structures of wild-type and mutant BbMetOx.

Main Results:

• Identified highly specific l-Met oxidizing LAAOs from Burkholderiales bacterium (BbMetOx) and Undibacterium sp. KW1 (UndMetOx).
• BbMetOx and UndMetOx exhibited significantly higher specific activity towards l-Met compared to other l-AAs.
• Optimal activity for these LAAOs was observed at pH 7.0 and 58-60°C.
• Crystal structures revealed conformational changes in the catalytic domain and identified four key residues (Y180, M182, F300, M302) crucial for l-Met recognition.

Conclusions:

• Sequence similarity network analysis is effective for discovering novel LAAOs with unique substrate specificities.
• The study advances understanding of LAAO enzyme family substrate specificity, particularly for alkyl-chain amino acids.
• Identified BbMetOx and UndMetOx represent valuable tools for biochemical research and potential biotechnological applications.