Molecular and structural basis for Cγ-C bond formation by PLP-dependent enzyme Fub7.

Publication Type:

Journal Article


Angew Chem Int Ed Engl, p.e202317161 (2024)


<p>Pyridoxal 5&#39;-phosphate (PLP)-dependent enzymes that catalyze γ-replacement reactions are prevalent, yet&nbsp;their utilization of carbon nucleophile substrates is rare. The recent discovery of two PLP-dependent enzymes,&nbsp;CndF and Fub7, has unveiled unique&nbsp;C-C bond forming capabilities, enabling the biocatalytic synthesis of&nbsp;alkyl-substituted pipecolic acids from O-acetyl-L-homoserine and β-keto acid or aldehyde derived enolates. This breakthrough presents fresh avenues for the biosynthesis of&nbsp;pipecolic acid derivatives.&nbsp;However, the&nbsp;catalytic mechanisms of these enzymes remain elusive, and a dearth of&nbsp;structural information hampers&nbsp;their extensive&nbsp;application. Here, we have broadened the catalytic scope of&nbsp;Fub7 by employing&nbsp;ketone-derived enolate&nbsp;as carbon nucleophiles, revealing&nbsp;Fub7&#39;s capacity for substrate-dependent regioselective α-alkylation of unsymmetrical ketones. Through an integrated approach combining&nbsp;X-ray crystallography, spectroscopy, mutagenesis, and computational docking studies, we offer&nbsp;a&nbsp;detailed mechanistic insight&nbsp;into&nbsp;Fub7 catalysis. Our findings elucidate the structural basis for its&nbsp;substrate specificity, stereoselectivity, and regioselectivity. Our work sets the stage ready for subsequent protein engineering effort aimed at expanding the synthetic utility of Fub7, potentially unlocking novel methods&nbsp;to access a broader array of noncanonical amino acids.</p>