Pabarcus Michael K, Casida John E
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA.
Biochim Biophys Acta. 2002 Apr 29;1596(2):201-11. doi: 10.1016/s0167-4838(02)00232-7.
Kynurenine formamidase (KFase) (EC 3.5.1.9) hydrolyzes N-formyl-L-kynurenine, an obligatory step in the conversion of tryptophan to nicotinic acid. Low KFase activity in chicken embryos, from inhibition by organophosphorus insecticides and their metabolites such as diazoxon, leads to marked developmental abnormalities. While KFase was purportedly isolated previously, the structure and residues important for catalysis and inhibition were not established. KFase was isolated here from mouse liver cytosol by (NH4)2SO4 precipitation and three FPLC steps (resulting in 221-fold increase in specific activity for N-formyl-L-kynurenine hydrolysis) followed by conversion to [3H]diethylphosphoryl-KFase and finally isolation by C4 reverse-phase high-performance liquid chromatography. Determination of tryptic fragment amino acid sequences and cDNA cloning produced a new 305-amino-acid protein sequence. Although an amidase by function, the primary structure of KFase lacks the amidase signature sequence and is more similar to esterases and lipases. Sequence profile analysis indicates KFase is related to the esterase/lipase/thioesterase family containing the conserved active-site serine sequence GXSXG. The alpha/beta-hydrolase fold is suggested for KFase by its primary sequence and predicted secondary conformation. A three-dimensional model based on the structures of homologous carboxylesterase EST2 and brefeldin A esterase implicates Ser162, Asp247 and His279 as the active site triad.
犬尿氨酸甲酰胺酶（KFase）（EC 3.5.1.9）可水解N-甲酰-L-犬尿氨酸，这是色氨酸转化为烟酸过程中的一个必要步骤。鸡胚中KFase活性较低，是由有机磷杀虫剂及其代谢产物如对氧磷的抑制作用导致的，这会引发明显的发育异常。虽然此前据称已分离出KFase，但对于催化和抑制作用至关重要的结构和残基尚未确定。在此，通过硫酸铵沉淀和三步快速蛋白质液相色谱法（FPLC）从小鼠肝脏胞质溶胶中分离出KFase（使N-甲酰-L-犬尿氨酸水解的比活性提高了221倍），随后将其转化为[3H]二乙基磷酰-KFase，最后通过C4反相高效液相色谱法进行分离。通过测定胰蛋白酶片段的氨基酸序列和进行cDNA克隆，得到了一个新的305个氨基酸的蛋白质序列。尽管KFase在功能上是一种酰胺酶，但其一级结构缺乏酰胺酶特征序列，与酯酶和脂肪酶更为相似。序列谱分析表明，KFase与包含保守活性位点丝氨酸序列GXSXG的酯酶/脂肪酶/硫酯酶家族相关。根据其一级序列和预测的二级构象，推测KFase具有α/β-水解酶折叠结构。基于同源羧酸酯酶EST2和布雷菲德菌素A酯酶的结构建立的三维模型表明，Ser162、Asp247和His279是活性位点三联体。
