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<p>Carbon-carbon bond formation is one of the key pillars of organic synthesis. Green, selective and efficient biocatalytic methods for such are therefore highly desirable. The α-oxoamine synthases (AOSs) are a class of pyridoxal 5’-phosphate (PLP)-dependent, irreversible, carbon-carbon bond-forming enzymes, which have been limited previously by their narrow substrate specificity and requirement of acyl-CoA thioester substrates. We recently characterized a thermophilic enzyme from <i>Thermus thermophilus</i> (<i>Th</i>AOS) with a much broader substrate scope and described its use in a chemo-biocatalytic cascade process to generate pyrroles in good yields and timescales. Herein, we report the structure-guided engineering of <i>Th</i>AOS to arrive at variants able to use a greatly expanded range of amino acid and simplified N-acetylcysteamine (SNAc) acyl-thioester substrates. The crystal structure of the improved <i>Th</i>AOS V79A variant with a bound PLP:<span>l</span>-penicillamine external aldimine ligand, provides insight into the properties of the engineered biocatalyst.</p>

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Rational engineering of a thermostable α-oxoamine synthase biocatalyst expands the substrate scope and synthetic applicability

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年3月13日

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  • Ben Ashley,
  • Sam Mathew,
  • Mariyah Sajjad,
  • Yaoyi Zhu,
  • Nikita Novikovs,
  • Arnaud Baslé,
  • Jon Marles-Wright,
  • Dominic J. Campopiano

摘要

Carbon-carbon bond formation is one of the key pillars of organic synthesis. Green, selective and efficient biocatalytic methods for such are therefore highly desirable. The α-oxoamine synthases (AOSs) are a class of pyridoxal 5’-phosphate (PLP)-dependent, irreversible, carbon-carbon bond-forming enzymes, which have been limited previously by their narrow substrate specificity and requirement of acyl-CoA thioester substrates. We recently characterized a thermophilic enzyme from Thermus thermophilus (ThAOS) with a much broader substrate scope and described its use in a chemo-biocatalytic cascade process to generate pyrroles in good yields and timescales. Herein, we report the structure-guided engineering of ThAOS to arrive at variants able to use a greatly expanded range of amino acid and simplified N-acetylcysteamine (SNAc) acyl-thioester substrates. The crystal structure of the improved ThAOS V79A variant with a bound PLP:l-penicillamine external aldimine ligand, provides insight into the properties of the engineered biocatalyst.