Journal of Biochemistry Advance Access originally published online on January 18, 2007
Journal of Biochemistry 2007 141(3):363-376; doi:10.1093/jb/mvm041
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2007 The Japanese Biochemical Society.
Functional and Structural Characterization of D-Aspartate Oxidase from Porcine Kidney: Non-Michaelis Kinetics due to Substrate Activation
Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-2192, Japan
*To whom correspondence should be addressed. Tel: +81 77 548 2158, Fax: +81 77 548 2157, E-mail: atsu{at}belle.shiga-med.ac.jp
Received December 25, 2006; Accepted January 9, 2007
 |
Abstract |
|---|
D-Aspartate oxidase (DDO, EC 1.4.3.1 [EC] ) catalyzes dehydrogenation of D-aspartate to iminoaspartate and the subsequent re-oxidation of reduced FAD with O2 to produce hydrogen peroxide. In the mammalian neuroendocrine system, D-aspartate, a natural substrate, plays important roles in the regulation of the synthesis and secretion of hormones. To elucidate the kinetic and structural properties of native DDO, we purified DDO from porcine kidney to homogeneity, cloned the cDNA, and overexpressed the enzyme in Escherichia coli. The purified DDO was a homotetramer with tightly-bound FAD. The enzyme consisted of 341 amino acids and had GAGVMG as the dinucleotide binding motif and a C-terminal SKL peroxisomal-targeting signal sequence. Porcine DDO showed a strong affinity for meso-tartrate (Kd = 118 µM). The oxidase exhibited pronounced substrate activation at D-aspartate and D-glutamate concentrations, [S], higher than 0.2 and 4 mM, respectively, and the [S]/v versus [S] plot showed marked downward curvature (v, the initial velocity), whereas substrate inhibition occurred with N-methyl-D-aspartate. These kinetic properties of DDO suggested that at high substrate concentrations, the FAD-reduced form of the enzyme also catalyzes the reaction: the oxidative half-reaction precedes the reductive one. The present direct approach to the analysis of non-Michaelis kinetics is indispensable for understanding the functional properties of DDO.
Key Words: D-aspartate oxidase, D-aspartate, cDNA cloning, neuroendocrine system, porcine kidney
Abbreviations: DDO, D-aspartate oxidase; NMDA, N-methyl-D-aspartic acid; MBTH, 3-methyl-2-benzothiazolinone hydrazone hydrochloride; RACE, rapid amplification of cDNA ends
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Ohnishi, M. Saito, S. Wakabayashi, M. Ishizuka, K. Nishimura, Y. Nagata, and S. Kasai Purification and Characterization of Serine Racemase from a Hyperthermophilic Archaeon, Pyrobaculum islandicum J. Bacteriol., February 15, 2008; 190(4): 1359 - 1365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tanaka, A. Yamamoto, T. Ishida, and K. Horiike D-Serine Dehydratase from Chicken Kidney: A Vertebral Homologue of the Cryptic Enzyme from Burkholderia cepacia J. Biochem., January 1, 2008; 143(1): 49 - 57. [Abstract] [Full Text] [PDF]
|
|