{'fraction_non_alphanumeric': 0.02976190476190476, 'fraction_numerical': 0.009424603174603174, 'mean_word_length': 5.634868421052632, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Emergent Decarboxylase Activity and Attenuation of α/β-Hydrolase Activity during the Evolution of Methylketone Biosynthesis in Tomato[C][W][OA]

Methylketones are insecticidal compounds that accumulate in certain plants as defense against herbivorous pests. Methylketone biosynthesis is a two-step process within the plant, with the second step, decarboxylation of 3-keto acids, catalyzed by Methylketone Synthase1 (MKS1). This study focuses on understanding the enzymatic mechanism of MKS1, which differs from proteins of the same structural class. Specialized methylketone-containing metabolites accumulate in certain plants, in particular wild tomatoes in which they serve as toxic compounds against chewing insects. In Solanum habrochaites f. glabratum, methylketone biosynthesis occurs in the plastids of glandular trichomes and begins with intermediates of de novo fatty acid synthesis. These fatty-acyl intermediates are converted via sequential reactions catalyzed by Methylketone Synthase2 (MKS2) and MKS1 to produce the n-1 methylketone. We report crystal structures of S. habrochaites MKS1, an atypical member of the α/β-hydrolase superfamily. Sequence comparisons revealed the MKS1 catalytic triad, Ala-His-Asn, as divergent to the traditional α/β-hydrolase triad, Ser-His-Asp. Determination of the MKS1 structure points to a novel enzymatic mechanism dependent upon residues Thr-18 and His-243, confirmed by biochemical assays. Structural analysis further reveals a tunnel leading from the active site consisting mostly of hydrophobic residues, an environment well suited for fatty-acyl chain binding. We confirmed the importance of this substrate binding mode by substituting several amino acids leading to an alteration in the acyl-chain length preference of MKS1. Furthermore, we employ structure-guided mutagenesis and functional assays to demonstrate that MKS1, unlike enzymes from this hydrolase superfamily, is not an efficient hydrolase but instead catalyzes the decarboxylation of 3-keto acids.