Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin, 53706, USA.
Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin, 53706, USA.
Chembiochem. 2022 Jul 19;23(14):e202200157. doi: 10.1002/cbic.202200157. Epub 2022 Jun 1.
Pyridoxal-5'-phosphate (PLP)-dependent enzymes have garnered interest for their ability to synthesize non-standard amino acids (nsAAs). One such class of enzymes, O-acetylserine sulfhydrylases (OASSs), catalyzes the final step in the biosynthesis of l-cysteine. Here, we examine the β-substitution capability of the OASS from Citrullus vulgaris (CvOASS), a putative l-mimosine synthase. While the previously reported mimosine synthase activity was not reproducible in our hands, we successfully identified non-native reactivity with a variety of O-nucleophiles. Optimization of reaction conditions for carboxylate and phenolate substrates led to distinct conditions that were leveraged for the preparative-scale synthesis of nsAAs. We further show this enzyme is capable of C-C bond formation through a β-alkylation reaction with an activated nitroalkane. To facilitate understanding of this enzyme, we determined the crystal structure of the enzyme bound to PLP as the internal aldimine at 1.55 Å, revealing key features of the active site and providing information that may guide subsequent development of CvOASS as a practical biocatalyst.
吡哆醛-5'-磷酸（PLP）依赖性酶因其能够合成非标准氨基酸（nsAAs）而引起了人们的兴趣。其中一类酶，O-乙酰丝氨酸硫基转移酶（OASSs），催化 l-半胱氨酸生物合成的最后一步。在这里，我们研究了来自葫芦科苦瓜（CvOASS）的 OASS 的β-取代能力，这是一种假定的 l-帽莫宁合酶。虽然我们手中以前报道的帽莫宁合酶活性不可重复，但我们成功地识别了与各种 O-亲核试剂的非天然反应性。优化羧酸盐和酚盐底物的反应条件导致了独特的条件，这些条件被用于 nsAAs 的制备规模合成。我们进一步表明，该酶能够通过与活化的硝基烷烃的β-烷基化反应形成 C-C 键。为了便于理解该酶，我们确定了该酶与 PLP 结合的晶体结构作为内部亚胺，分辨率为 1.55 Å，揭示了活性位点的关键特征，并提供了可能指导随后将 CvOASS 开发为实用生物催化剂的信息。
