Studies on Phe-228 and Leu-307 Recombinant Mutants of Porcine Kidney D-Amino Acid Oxidase: Expression, Purification, and Characterization1

Abstract

Two recombinant mutants of porcine kidney D-amino acid oxidase [EC 1.4.3.3, DAO], in which Tyr(228) and His(307) are replaced with Phe and Leu, respectively, have been expressed in Escherichia coli and purified to apparent homogeneity. The molecular size and amino-terminal sequence of the two mutants were the same as those of the native DAO. Kinetic analysis revealed that the Michaelis constants of the Phe-228 and Leu-307 mutants for D-alanine were 71- and 10-fold and the inhibition constants for benzoate, a potent competitive inhibitor, were 1,189- and 18-fold greater than those of the native DAO, respectively. The maximum velocities of the Phe-228 and Leu-307 mutants were 66 and 58% that of the native DAO. The kinetically estimated dissociation constant of the Leu-307 mutant for FAD was 28-fold greater than that of the native DAO, whereas the value of the Phe-228 mutant was comparable to that of the native DAO. The Leu-307 mutant and the recombinant wild-type DAO were inactivated by D-propargylglycine (D-PG), a suicide substrate. However, the Phe-228 mutant was resistant to the inactivation. Absorption peaks of the Phe-228 mutant were blue-shifted about 10 nm from the corresponding peaks of the wild-type DAO, and the oxidized form was fully reduced by D-alanine without appearance of the purple intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorpromazine oligomer is a potentially active substance that inhibits humanD-amino acid oxidase, product of a susceptibility gene for schizophrenia

“…The following values were obtained at pH 8.3 and 258C from doublereciprocal plots: Michaelis constant K m (for Dalanine) ¼ 1 mM, the apparent dissociation constant for FAD K f ¼ 0.2 mM. These values are in good agreement with those reported [24,29]. The apoprotein used was found to have no activity without the addition of exogenous FAD.…”

“…Each reaction time was less than approximately 10 min. Kinetic parameters of the enzyme reactions were calculated from the conventional Michaelis equations [29,30]. The following values were obtained at pH 8.3 and 258C from doublereciprocal plots: Michaelis constant K m (for Dalanine) ¼ 1 mM, the apparent dissociation constant for FAD K f ¼ 0.2 mM.…”

Glycerol-assisted Restorative Adjustment of Flavoenzyme Conformation Perturbed by Site-directed Mutagenesis

“…Cell Growth and Purification of DAAO-Escherichia coli cells transformed with the expression plasmid that contain wild type or H307L-mutated DAAO cDNA (15,18) were generous gifts of Dr. Kiyoshi Fukui. The cell culture growth and crude extract preparation were performed according to Pollegioni et al (19).…”

“…His-307 is one of two residues (the other is Tyr-228) undergoing affinity labeling with the D-propargylglycine-suicide substrate of pig kidney DAAO (11)(12)(13). Furthermore, the replacement of His-307 with Leu greatly reduces the protein affinity for FAD so that DAAO can be isolated only as an inactive apoprotein (14,15). Although this is an indication that the presence of His-307 is important for protein-flavin interactions, the role of this residue remains vague (15).…”

“…Furthermore, the replacement of His-307 with Leu greatly reduces the protein affinity for FAD so that DAAO can be isolated only as an inactive apoprotein (14,15). Although this is an indication that the presence of His-307 is important for protein-flavin interactions, the role of this residue remains vague (15). The recently determined crystal structure of pig kidney DAAO shows that although His-307 is located in the flavin binding domain it is not closely positioned toward FAD (16,17).…”