Department of Life Sciences and Institute of Genome Sciences, National Yang Ming Chiao Tung University, Taipei, Taiwan; Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan.
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan.
Colloids Surf B Biointerfaces. 2022 Feb;210:112224. doi: 10.1016/j.colsurfb.2021.112224. Epub 2021 Nov 16.
Pyridoxal phosphate (PLP)-dependent α-oxoamine synthases are generally believed to be responsible for offloading and elongating polyketides or catalyzing the condensation of amino acids and acyl-CoA thioester substrates, such as serine into sphingolipids and cysteate into sulfonolipids. Previously, we discovered vitroprocines, which are tyrosine- and phenylalanine-polyketide derivatives, as potential new antibiotics from the genus Vibrio. Using bioinformatics analysis, we identified putative genes of PLP-dependent enzyme from marine Vibrio sp. QWI-06, implying a capability to produce amino-polyketide derivatives. One of these genes was cloned, and the recombinant protein, termed Vibrio sp. QWI-06 α-oxoamine synthases-1 (VsAOS1), was overexpressed for structural and biochemical characterization. The crystal structure of the dimeric VsAOS1 was determined at 1.8-Å resolution in the presence of L-glycine. The electron density map indicated a glycine molecule occupying the pyridoxal binding site in one monomer, suggesting a snapshot of the initiation process upon the loading of amino acid substrate. In mass spectrometry analysis, VsAOS1 strictly acted to condense L-glycine with C12 or C16 acyl-CoA, including unsaturated acyl analog. Furthermore, a single residue replacement of VsAOS1 (G243S) allowed the enzyme to generate sphingoid derivative when L-serine and lauroyl-CoA were used as substrates. Our data elucidate the mechanism of substrate binding and selectivity by the VsAOS1 and provide a thorough understanding of the molecular basis for the amino acid preference of AOS members.
吡哆醛磷酸 (PLP) 依赖的α-氧胺合酶通常被认为负责卸载和延长聚酮或催化氨基酸和酰基辅酶 A 硫酯底物的缩合，如丝氨酸成鞘脂和半胱氨酸成磺酰脂。以前，我们从弧菌属发现了维罗普罗辛，它是酪氨酸和苯丙氨酸聚酮衍生物，是潜在的新型抗生素。使用生物信息学分析，我们从海洋弧菌 sp. QWI-06 中鉴定出了 PLP 依赖酶的假定基因，这表明它们有产生氨基酸聚酮衍生物的能力。其中一个基因被克隆，重组蛋白，称为弧菌 sp. QWI-06 α-氧胺合酶-1 (VsAOS1)，被过度表达用于结构和生化特性分析。在存在 L-甘氨酸的情况下，二聚体 VsAOS1 的晶体结构在 1.8-Å 分辨率下被确定。电子密度图表明一个甘氨酸分子占据了一个单体中吡哆醛结合位点，这表明在氨基酸底物加载时的起始过程的一个快照。在质谱分析中，VsAOS1 严格地将 L-甘氨酸与 C12 或 C16 酰基辅酶 A，包括不饱和酰基类似物缩合。此外，当 L-丝氨酸和月桂酰辅酶 A 用作底物时，VsAOS1 的单个残基替换 (G243S) 允许酶生成神经酰胺衍生物。我们的数据阐明了 VsAOS1 底物结合和选择性的机制，并提供了对 AOS 成员对氨基酸偏好的分子基础的全面理解。
