Ebselen: Mechanisms of Glutamate Dehydrogenase and Glutaminase Enzyme Inhibition.

Ebselen modulates target proteins through redox reactions with selenocysteine/cysteine residues, or through binding to the zinc finger domains. However, a recent contradiction in ebselen inhibition of kidney type glutaminase (KGA) stimulated our interest in investigating its inhibition mechanism with glutamate dehydrogenase (GDH), KGA, thioredoxin reductase (TrxR), and glutathione S-transferase. Fluorescein- or biotin-labeled ebselen derivatives were synthesized for mechanistic analyses. Biomolecular interaction analyses showed that only GDH, KGA, and TrxR proteins can bind to the ebselen derivative, and the binding to GDH and KGA could be competed off by glutamine or glutamate. From the gel shift assays, the fluorescein-labeled ebselen derivative could co-migrate with hexameric GDH and monomeric/dimeric TrxR in a dose-dependent manner; it also co-migrated with KGA but disrupted the tetrameric form of the KGA enzyme at a high compound concentration. Further proteomic analysis demonstrated that the ebselen derivative could cross-link with proteins through a specific cysteine at the active site of GDH and TrxR proteins, but for KGA protein, the binding site is at the N-terminal appendix domain outside of the catalytic domain, which might explain why ebselen is not a potent KGA enzyme inhibitor in functional assays. In conclusion, ebselen could inhibit enzyme activity by binding to the catalytic domain or disruption of the protein complex. In addition, ebselen is a relatively potent selective GDH inhibitor that might provide potential therapeutic opportunities for hyperinsulinism-hyperammonemia syndrome patients who have the mutational loss of GTP inhibition.

A new class of glutaminase from Aspergillus oryzae.

The koji mold Aspergillus oryzae is able to produce glutaminase which converts glutamine to glutamic acid, one of the most important flavor components in soy sauce. We present here the isolation and the complete nucleotide sequence of the glutaminase- encoding gene from A. oryzae U212, an industrial strain used in Thailand. N-terminal and internal amino acid sequences were determined from purified glutaminase. A 700-bp fragment was amplified by PCR using oligonucleotide primers designed from partial amino acid sequences. This PCR fragment was used as a homologous probe for screening an A. oryzae genomic DNA library. RT-PCR showed that the gene contained seven short introns. Sequence analysis revealed an open reading frame that encodes a protein of 690 amino-acid residues with a predicted molecular mass of 76 kDa. The N-terminal and internal amino acid sequences of the deduced protein exactly matched the ones determined from the purified protein. Comparison of the amino acid sequence with glutaminase sequences from other origins showed that A. oryzae glutaminase shares little homology with those of bacteria, eukaryote and mammals. The A. oryzae glutaminase gene was expressed in A. nidulans to confirm the presence of a functional glutaminase gene in the cloned DNA. To our knowledge, this is the first reported glutaminase gene cloned from filamentous fungi.