Structural and Mechanistic Basis of Bacterial 2-Methyl-ACC Synthases.

Cyclopropane-containing amino acids are found in diverse natural products and contribute to their structural complexity and biological activity. However, the distribution of bacterial biosynthetic pathways that produce methylated cyclopropane amino acids and the structural basis of methyl-SAM recognition remain poorly understood. Here, we show that pyridoxal 5'-phosphate (PLP)-dependent 1-amino-2-methylcyclopropane-1-carboxylic acid (2-methyl-ACC) synthases are widely distributed in bacteria. Although Orf30 preferentially converts (R)-4″-methyl-SAM into (1S,2S)-methyl-ACC, some homologues also accept the (S)-4″-methyl-SAM stereoisomer. Structural, computational, and mutational analyses of Orf30, the PLP-dependent enzyme responsible for formation of the 2-methyl-ACC moiety in the natural product Q6402A, suggest that the shape and hydrophobicity of the C4″-methyl-binding region contribute to productive substrate positioning and stereochemical preference. Molecular dynamics simulations, density functional theory calculations, and mutational analyses further support direct cyclopropane formation from a PLP-bound quinonoid intermediate without requiring a substrate intermediate covalently bound to the enzyme at C4″. Together, these findings show how active-site architecture couples substrate stereochemical recognition with quinonoid-driven cyclopropanation, expanding our understanding of ACC synthase-like PLP enzymes.