Cloning, expression, and catalytic triad of recombinant arylformamidase.
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.
Pabarcus
Michael K
MK
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA.
Casida
John E
JE
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D005558:Arylformamidase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D008099:Liver / Q000201:enzymology*; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000379:methods; D017354:Point Mutation / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D005558:Arylformamidase