Momany Cory, Levdikov Vladimir, Blagova Lena, Lima Santiago, Phillips Robert S
Department of Chemistry, University of Georgia, Athens, Georgia 30602, USA.
Biochemistry. 2004 Feb 10;43(5):1193-203. doi: 10.1021/bi035744e.
Kynureninase [E.C. 3.7.1.3] is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the hydrolytic cleavage of l-kynurenine to anthranilic acid and l-alanine. Sequence alignment with other PLP-dependent enzymes indicated that kynureninase is in subgroup IVa of the aminotransferases, along with nifS, CsdB, and serine-pyruvate aminotransferase, which suggests that kynureninase has an aminotransferase fold. Crystals of Pseudomonas fluorescens kynureninase were obtained, and the structure was solved by molecular replacement using the CsdB coordinates combined with multiple isomorphous heavy atom replacement. The coordinates were deposited in the PDB (ID code 1QZ9). The structure, refined to an R factor of 15.5% to 1.85 A resolution, is dimeric and has the aminotransferase fold. The structure also confirms the prediction from sequence alignment that Lys-227 is the PLP-binding residue in P. fluorescens kynureninase. The conserved Asp-201, expected for an aminotransferase fold, is located near the PLP nitrogen, but Asp-132 is also strictly conserved and at a similar distance from the pyridinium nitrogen. Mutagenesis of both conserved aspartic acids shows that both contribute equally to PLP binding, but Asp-201 has a greater role in catalysis. The structure shows that Tyr-226 donates a hydrogen bond to the phosphate of PLP. Unusual among PLP-dependent enzymes, Trp-256, which is also strictly conserved in kynureninases from bacteria to humans, donates a hydrogen bond to the phosphate through the indole N1-hydrogen.
犬尿氨酸酶[E.C. 3.7.1.3]是一种依赖于磷酸吡哆醛（PLP）的酶，催化L-犬尿氨酸水解裂解生成邻氨基苯甲酸和L-丙氨酸。与其他依赖PLP的酶进行序列比对表明，犬尿氨酸酶与nifS、CsdB和丝氨酸-丙酮酸转氨酶一起属于转氨酶的IVa亚组，这表明犬尿氨酸酶具有转氨酶折叠结构。获得了荧光假单胞菌犬尿氨酸酶的晶体，并通过使用CsdB坐标结合多同晶型重原子置换的分子置换法解析了其结构。这些坐标已存入蛋白质数据银行（PDB，ID代码1QZ9）。该结构在1.85 Å分辨率下精修至R因子为15.5%，为二聚体，具有转氨酶折叠结构。该结构还证实了序列比对的预测结果，即Lys-227是荧光假单胞菌犬尿氨酸酶中的PLP结合残基。对于转氨酶折叠结构预期保守的Asp-201位于PLP氮附近，但Asp-132也严格保守，且与吡啶鎓氮的距离相似。对两个保守天冬氨酸进行诱变表明，二者对PLP结合的贡献相同，但Asp-201在催化中起更大作用。该结构表明Tyr-226向PLP的磷酸基团提供氢键。在依赖PLP的酶中不同寻常的是，Trp-256（从细菌到人类的犬尿氨酸酶中也严格保守）通过吲哚N1-氢向磷酸基团提供氢键。
