Molecular and Structural Basis for Cγ-C Bond Formation by PLP-Dependent Enzyme Fub7

Area of Science:

• Biocatalysis and Enzyme Engineering
• Organic Chemistry
• Structural Biology

Background:

• Pyridoxal 5"-phosphate (PLP)-dependent enzymes typically catalyze γ-replacement reactions, with rare use of carbon nucleophiles.
• Recent discoveries of CndF and Fub7 enzymes show potential for C-C bond formation in synthesizing alkyl-substituted pipecolic acids.

Purpose of the Study:

• To expand the catalytic scope of the Fub7 enzyme using ketone-derived enolates.
• To elucidate the catalytic mechanism, substrate specificity, stereoselectivity, and regioselectivity of Fub7.
• To provide structural insights for future protein engineering of Fub7.

Main Methods:

• Broadened Fub7's catalytic scope using ketone-derived enolates.
• Integrated X-ray crystallography, spectroscopy, mutagenesis, and computational docking.
• Analyzed substrate-dependent regioselective α-alkylation of unsymmetrical ketones.

Main Results:

• Demonstrated Fub7's capability for regioselective α-alkylation of unsymmetrical ketones with ketone-derived enolates.
• Provided detailed mechanistic insights into Fub7 catalysis.
• Elucidated the structural basis for Fub7's substrate specificity, stereoselectivity, and regioselectivity.

Conclusions:

• Fub7 exhibits broader substrate acceptance and regioselective C-C bond formation capabilities than previously known.
• Structural and mechanistic understanding paves the way for protein engineering to enhance Fub7's synthetic utility.
• Potential for novel biocatalytic methods to synthesize diverse noncanonical amino acids.