Hedges Jason B, Marchand Jorge A, Calvó-Tusell Carla, Wei Zi-Wang, Millar Douglas C, Garcia-Borràs Marc, Chang Michelle C Y, Ryan Katherine S
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada.
Xilio Therapeutics, Waltham, MA, USA.
Nat Chem Biol. 2025 Jun 25. doi: 10.1038/s41589-025-01954-9.
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.
含末端炔烃的天然产物可进行铜（I）催化的叠氮化物-炔烃环加成这一生物正交“点击”反应。最近，在L-β-乙炔基丝氨酸的生物合成过程中发现了一种形成末端炔烃的酶促机制，其中依赖磷酸吡哆醛的酶BesB从卤代乙烯前体4-氯-L-烯丙基甘氨酸形成了一种罕见的含末端炔烃的氨基酸L-炔丙基甘氨酸。在此，我们展示了BesB分辨率为1.3 Å的晶体结构，并进行了详细的机制和计算研究。我们证明BesB可以可逆地催化各种4-卤代烯丙基-L-甘氨酸中卤素的交换，这意味着存在一个丙二烯中间体，我们随后也观察到了该中间体。综上所述，这项工作支持了一种机制，即通过去质子化驱动的卤素损失形成丙二烯，然后通过丙二烯的异构化形成末端炔烃。我们的工作进一步扩展了我们对依赖磷酸吡哆醛的酶的催化功能的理解，并将推动无金属的丙二烯形成和炔烃形成生物催化剂的开发。
