Abstract：
The Gram-negative bacterium, Fusobacterium nucleatum, possesses a fold II type pyridoxal 5\'-phosphate-dependent enzyme that catalyzes the reversible β-replacement of l-cysteine and l-serine, generating H2S and H2O, respectively. This enzyme, termed serine synthase (FN1055), contains an active site Asp232 that serves as a general base in the activation of a water molecule for nucleophilic attack of the ⍺-aminoacrylate intermediate. A network of hydrophobic residues surrounding Asp232 are key to catalysis as they increase the basicity of the side chain. However, these residues severely restrict the range of nucleophilic substrates that can react with the ⍺-aminoacrylate, making the enzyme an ineffective biocatalyst for noncanonical amino acid biosynthesis. Herein, we systematically substituted four aromatic active residues (Trp99, Phe125, Phe148 and Phe234) to an alanine to determine their catalytic importance in serine/cysteine synthase reactions and if their substitution could broaden the scope of nucleophiles that could react with the ⍺-aminoacrylate intermediate. All four single site mutants W99A, F125A, F148A, and F234A could form the ⍺-aminoacrylate intermediate upon reaction with either l-cysteine or l-serine; however, the rate constant associated with the elimination of the β-hydroxyl group from l-serine was 150 to 200-fold lower in the F125A and F148A variants. Substitution of Phe125 and Phe148, situated ∼3-4 Å from the general base, also abolished the serine synthase reaction due to their inability to activate a water molecule for nucleophilic attack of the ⍺-aminoacrylate. Overall, the mutational studies indicate that the clustering of aromatic residues disproportionately benefits the serine synthase reaction as they increase the binding affinity for l-cysteine, decrease the binding of the product, l-serine, and promote the activation of a water molecule. Notably, the aminoacrylate species present in F125A and F148A was able to react with thiophenol, signifying that serine synthase has biocatalytic potential in the synthesis of noncanonical amino acids.
摘要：
革兰氏阴性细菌，具核梭杆菌,具有折叠II型吡哆醛5'-磷酸依赖性酶，可催化1-半胱氨酸和1-丝氨酸的可逆β-置换，产生H2S和H2O，分别。这种酶，称为丝氨酸合酶（FN1055），含有活性位点Asp232，该活性位点在水分子的活化中用作一般碱，用于亲核攻击β-氨基丙烯酸酯中间体。围绕Asp232的疏水残基网络是催化的关键,因为它们增加侧链的碱性。然而,这些残基严重限制了可以与β-氨基丙烯酸酯反应的亲核底物的范围，使酶成为非规范氨基酸生物合成的无效生物催化剂。在这里，我们系统地将四个芳香族活性残基（Trp99，Phe125，Phe148和Phe234）取代为丙氨酸，以确定它们在丝氨酸/半胱氨酸合酶反应中的催化重要性，以及它们的取代是否可以扩大可以与-氨基丙烯酸酯中间体反应的亲核试剂的范围。所有四个单位点突变体W99A，F125A,F148A,和F234A可以在与1-半胱氨酸或1-丝氨酸反应后形成α-氨基丙烯酸酯中间体；然而，在F125A和F148A变体中,与从1-丝氨酸中消除β-羟基相关的速率常数低150~200倍.Phe125和Phe148的替代，位于一般基地的3-4，也取消了丝氨酸合酶反应，因为它们无法激活水分子以进行α-氨基丙烯酸酯的亲核攻击。总的来说,突变研究表明，芳香残基的聚类不成比例地有利于丝氨酸合酶反应，因为它们增加了对l-半胱氨酸的结合亲和力，减少产品的结合，l-丝氨酸,并促进水分子的活化。值得注意的是,F125A和F148A中存在的氨基丙烯酸酯能够与苯硫酚反应，表明丝氨酸合酶在非规范氨基酸的合成中具有生物催化潜力。
