The role of residues Arg169 and Arg220 in intersubunit interactions of yeast D-amino acid oxidase

Abstract

D Amino acid oxidase from the yeast Trigonopsis variabilis (EC 1.4.3.3, TvDAAO) exists as a dimer consisting of two identical subunits. The dimeric structure of the enzyme is stabilized by 12 (six pairs) hydrogen bonds, the residues Arg169 and Arg220 of each subunit being involved in eight hydrogen bonds. The Arg169Glu and Arg(169,220)Ala mutants of TvDAAO were pre pared. Both mutant enzymes were expressed in E. coli cells as insoluble but catalytically active inclusion bodies. The introduction of amino acid substitutions at the intersubunit interface resulted in a change in the substrate specificity profile and a strong decrease in thermal stability.Key words: D amino acid oxidase, site directed mutagenesis, quaternary structure, sub strate specificity, thermal stability. D Amino acid oxidase (EC 1.4.3.3, DAAO) is the FAD containing enzyme that catalyzes the oxidative deamination of D amino acids to form α keto acids, hy drogen peroxide, and an ammonium ion. In spite of the fact that the enzyme plays an essential role in the regula tion of a number of very important processes in animal organisms (nervous activity, hormone secretion, etc.) 1,2 and is widely used in practice, 3-5 the structure-function relationship of DAAOs from different sources is little known. By 2008, the structures of only three D amino acid oxidases were solved, viz., those from porcine kidney (pkDAAO), 6,7 human kidney (hDAAO), 8,9 and the yeast Rhodotorula gracilis (RgDAAO). 10,11 The enzyme from the yeast Trigonopsis variabilis (TvDAAO) is the fourth best characterized DAAO. The latter enzyme was obtained 12 in a highly purified state in 1976 and is most widely used in practice. 3-5 One can assume that, in view of the practical importance of this enzyme, numerous attempts were made in the last 30 years to crystallize TvDAAO. However, no structural data on this enzyme are available in the Protein Data Bank (PDB) and there are no publications concern ing this problem, which suggests that all the attempts were unsuccessful. We have also carried out a large scale screen ing of the crystallization conditions for wild type TvDAAO but failed to obtain crystals. Only after obtaining the Cys108Phe mutant of TvDAAO, 13 we succeeded in grow ing crystals of this enzyme and then solved its structure first at 2.8 Å resolution 14 and then at 1.8 Å resolution. 2 Porcine kidney D amino acid oxidase in vivo is a mono mer, whereas the enzymes from other sources exist in na ture as dimers. In the crystal, pkDAAO also exists as a dimer, in which two subunits are linked in a head to head fashion. 6,7 For human D amino acid oxidase, two crystal modifications were grown. In one modification, like in the dimer of pkDAAO, the subunits are arranged in a head to head fashion, 9 whereas in another modification the subunits are arranged in a cross like fashion. 8 The dimers of TvDAAO C108F and RgDAAO are arranged in a head to tail fashion. However, unlike the latter enzyme, the subunits of the TvDAAO C108F molecule do not lie in one plane and are shifted with respect...

Study of the Structure-Function-Stability Relationships in Yeast D-amino Acid Oxidase: Hydrophobization of Alpha-Helices

“…The Ser157 and Ser161 residues are located at the intersubunit area. Therefore, replacement of these residues is also undesirable, despite the fact that they do not participate in the formation of intersubunit hydrogen bonds [ 11 ]. Thus, eight Ser residues were selected to be replaced with Ala residues (positions 67, 77, 78, 105, 270, 277, 335, and 336).…”

“…We showed [ 11 , 13 , 16 ] that inactivation of wild-type TvDAAO and its various mutants at elevated temperatures proceeds according to the following dissociative mechanism:…”

“…[ 18 ]. The time dependence of the residual activity of the enzyme in this mechanism is described by a sum of two exponential functions, and the inactivation rate of the enzyme depends on its concentration [ 11 , 13 , 16 ]. The dissociative mechanism of the wild-type TvDAAO thermoinactivation is only observed within a temperature range of 50-60°C, when the rate constants k 1 and k 2 are comparable to each other.…”

“…In our laboratory, the D-amino acid oxidase gene from T. variabilis yeast has been cloned, the overexpression system of the recombinant enzyme in Escherichia coli cells in soluble and active form has been developed, and its properties have been studied [ 10 ]. Native TvDAAO is a homodimer [ 11 ], which has a 2-fold symmetry axis with the subunits mutually arranged in a “head-to-tail” manner. Each subunit contains one FAD cofactor molecule at the active site.…”