O'Hare Helen M, Huang Fanglu, Holding Andrew, Choroba Oliver W, Spencer Jonathan B
Department of Chemistry, University of Cambridge, UK.
FEBS Lett. 2006 Jun 12;580(14):3445-50. doi: 10.1016/j.febslet.2006.05.018. Epub 2006 May 15.
Hydroxymandelate synthase (HmaS) and hydroxyphenylpyruvate dioxygenase (HppD) are non-heme iron-dependent dioxygenases, which share a common substrate and first catalytic step. The catalytic pathways then diverge to yield hydroxymandelate for secondary metabolism, or homogentisate in tyrosine catabolism. To probe the differences between these related active sites that channel a common intermediate down alternative pathways, we attempted to interconvert their activities by directed evolution. HmaS activity was readily introduced to HppD by just two amino acid changes. A parallel attempt to engineer HppD activity in HmaS was unsuccessful, suggesting that homogentisate synthesis places greater chemical and steric demands on the active site.
羟基扁桃酸合酶（HmaS）和对羟基苯丙酮酸双加氧酶（HppD）是依赖非血红素铁的双加氧酶，它们共享一个共同的底物和第一步催化反应。然后催化途径发生分歧，产生用于次生代谢的羟基扁桃酸，或在酪氨酸分解代谢中产生尿黑酸。为了探究这些相关活性位点之间的差异，这些活性位点将一个共同的中间体导向不同的途径，我们试图通过定向进化来相互转换它们的活性。仅通过两个氨基酸的改变，HmaS的活性就很容易被引入到HppD中。在HmaS中设计HppD活性的平行尝试未成功，这表明尿黑酸的合成对活性位点提出了更大的化学和空间要求。
