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.
Protein Expr Purif. 2005 Nov;44(1):39-44. doi: 10.1016/j.pep.2005.04.013.
Arylformamidase (AFMID) is the second enzyme of the kynurenine pathway metabolizing tryptophan to nicotinic acid and nicotinamide adenine dinucleotide cofactors. Inhibition of AFMID by organophosphorus insecticides in developing chicken embryos is correlated with lowered NAD levels and severe teratogenesis. The cDNA sequence previously identified for mouse liver AFMID (AF399717) (MW 34229) was cloned and expressed in Escherichia coli. Residues identified as potential catalytic triad members (S162, D247, and H279) through sequence motif and homology modeling were mutated to alanine to probe their contributions to enzyme activity. The wild-type and mutant AFMIDs were expressed as amino terminal 6 x His-tagged recombinant proteins to facilitate purification. Three chromatography steps isolated highly purified proteins for enzyme activity comparisons. Expressed AFMID showed high activity, 42+/-1 micromol/min/mg protein, for its natural substrate, N-formyl-l-kynurenine. The same K(m) (0.18--0.19 mM) was observed for expressed and native cytosolic AFMID. The single mutants (S162A, D247A, and H279A) lost essentially all (>99%) activity. The predicted catalytic triad of S162, D247, and H279 is therefore confirmed by site-directed mutagenesis.
芳基甲酰胺酶（AFMID）是犬尿氨酸途径中的第二种酶，可将色氨酸代谢为烟酸和烟酰胺腺嘌呤二核苷酸辅因子。在发育中的鸡胚中，有机磷杀虫剂对AFMID的抑制作用与NAD水平降低和严重的致畸作用相关。先前鉴定的小鼠肝脏AFMID（AF399717）（分子量34229）的cDNA序列被克隆并在大肠杆菌中表达。通过序列基序和同源性建模确定为潜在催化三联体成员的残基（S162、D247和H279）被突变为丙氨酸，以探究它们对酶活性的贡献。野生型和突变型AFMID被表达为氨基末端带有6×组氨酸标签的重组蛋白，以利于纯化。通过三步色谱法分离出高度纯化的蛋白用于酶活性比较。表达的AFMID对其天然底物N-甲酰基-L-犬尿氨酸显示出高活性，为42±1微摩尔/分钟/毫克蛋白。表达的和天然胞质AFMID的K(m)值相同（0.18 - 0.19 mM）。单突变体（S162A、D247A和H279A）基本上丧失了所有（>99%）活性。因此，通过定点诱变证实了预测的S162、D247和H279催化三联体。
