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<p>Terminal alkyne-containing natural products can undergo the bio-orthogonal ‘click’ reaction of Cu(I)-catalyzed azide–alkyne cycloaddition. Recently, an enzymatic mechanism for terminal alkyne formation was discovered in the biosynthesis of <span>l</span>-β-ethynylserine where the pyridoxal phosphate-dependent enzyme BesB forms a rare terminal alkyne-containing amino acid, <span>l</span>-propargylglycine, from a vinyl halide precursor, 4-chloro-<span>l</span>-allylglycine. Here we present the 1.3-Å-resolution crystal structure of BesB with detailed mechanistic and computational studies. We demonstrate that BesB can reversibly catalyze the exchange of the halogen in various 4-halo-allyl-<span>l</span>-glycines, implying the existence of an allene intermediate, which we then also observe. Taken together, this work supports a mechanism whereby an allene is formed from deprotonation-driven halogen loss and the terminal alkyne is then formed by isomerization of the allene. Our work further expands our understanding of the catalytic repertoire of pyridoxal phosphate-dependent enzymes and will enable development of metal-free allene-forming and alkyne-forming biocatalysts.</p><p></p>

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Terminal alkyne formation by a pyridoxal phosphate-dependent enzyme

  • 期刊论文

  • 发表日期：

    2025年6月25日

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  • Jason B. Hedges,
  • Jorge A. Marchand,
  • Carla Calvó-Tusell,
  • Zi-Wang Wei,
  • Douglas C. Millar,
  • Marc Garcia-Borràs,
  • Michelle C. Y. Chang,
  • Katherine S. Ryan

摘要

Terminal alkyne-containing natural products can undergo the bio-orthogonal ‘click’ reaction of Cu(I)-catalyzed azide–alkyne cycloaddition. Recently, an enzymatic mechanism for terminal alkyne formation was discovered in the biosynthesis of l-β-ethynylserine where the pyridoxal phosphate-dependent enzyme BesB forms a rare terminal alkyne-containing amino acid, l-propargylglycine, from a vinyl halide precursor, 4-chloro-l-allylglycine. Here we present the 1.3-Å-resolution crystal structure of BesB with detailed mechanistic and computational studies. We demonstrate that BesB can reversibly catalyze the exchange of the halogen in various 4-halo-allyl-l-glycines, implying the existence of an allene intermediate, which we then also observe. Taken together, this work supports a mechanism whereby an allene is formed from deprotonation-driven halogen loss and the terminal alkyne is then formed by isomerization of the allene. Our work further expands our understanding of the catalytic repertoire of pyridoxal phosphate-dependent enzymes and will enable development of metal-free allene-forming and alkyne-forming biocatalysts.