Isolation and characterization of a D‐cysteine desulfhydrase protein from Arabidopsis thaliana

Abstract

It is well documented that, in general, amino acids are used in the l-form, and enzymes involved in their metabolism are stereospecific for the l-enantiomers. However, d-amino acids are widely distributed in living organisms [1]. Examples of the natural occurrence of d-amino acids include d-amino acid-containing natural peptide toxins [2], antibacterial diastereomeric peptides [3], and the presence of d-amino acids at high concentrations in human brain [4]. In plants d-amino acids were detected in gymnosperms as well as monoand dicotyledonous angiosperms of major plant families. Free d-amino acids in the low percentage range of 0.5-3% relative to their l-enantiomers are principle constituents of plants [5]. The functions of d-amino acids and their metabolism are largely unknown. Various pyridoxal-5¢-phosphate (PLP)-dependent enzymes that catalyse elimination and replacement reactions of amino acids have been purified and characterized [6]. In several organisms d-cysteine desulfhydrase (d-CDes) activity (EC 4.1.99.4) was measured; this enzyme decomposes d-cysteine into pyruvate, H 2 S, and NH 3 . A gene encoding a putative d-CDes protein was identified in Arabidopsis thaliana (L) Heynh. based on high homology to an Escherichia coli protein called YedO that has d-CDes activity. The deduced Arabidopsis protein consists of 401 amino acids and has a molecular mass of 43.9 kDa. It contains a pyridoxal-5¢-phosphate binding site. The purified recombinant mature protein had a K m for d-cysteine of 0.25 mm. Only d-cysteine but not l-cysteine was converted by d-CDes to pyruvate, H 2 S, and NH 3 . The activity was inhibited by aminooxy acetic acid and hydroxylamine, inhibitors specific for pyridoxal-5¢-phosphate dependent proteins, at low micromolar concentrations. The protein did not exhibit 1-aminocyclopropane-1-carboxylate deaminase activity (EC 3.5.99.7) as homologous bacterial proteins. Western blot analysis of isolated organelles and localization studies using fusion constructs with the green fluorescent protein indicated an intracellular localization of the nuclear encoded d-CDes protein in the mitochondria. d-CDes RNA levels increased with proceeding development of Arabidopsis but decreased in senescent plants; d-CDes protein levels remained almost unchanged in the same plants whereas specific d-CDes activity was highest in senescent plants. In plants grown in a 12-h light ⁄ 12-h dark rhythm d-CDes RNA levels were highest in the dark, whereas protein levels and enzyme activity were lower in the dark period than in the light indicating post-translational regulation. Plants grown under low sulfate concentration showed an accumulation of d-CDes RNA and increased protein levels, the d-CDes activity was almost unchanged. Putative in vivo functions of the Arabidopsis d-CDes protein are discussed.Abbreviations

Hydrogen sulfide is involved in the chilling stress response in Vitis vinifera L.

“…Similar to animals, the homologs of CBS (LCD) and CSE (DCD) also are found in plants, and they are reported mainly responsible for generating H 2 S [4,36]. LCD (At3g62130) and DCD (At1g48420) genes have been isolated from Arabidopsis [2,37].…”

Molecular Biology, Biochemistry and Cellular Physiology of Cysteine Metabolism inArabidopsis thaliana

“…CS-like null mutants exhibit decreased total cysteine desulfhydrase activity, indicating the presence of additional enzymes with similar activities (Ãlvarez et al, 2010a). Next, D-Cys desulfhydrases (EC 4.4.1.15) with high specificity for this enantionmer have been identified (Riemenschneider et al, 2005;Papenbrock et al, 2007). This reaction would be important in combination with a cysteine amino-acid racemase which still needs to be demonstrated (Gördes et al, 2010), leaving the role of D-cysteine unclear (Papenbrock et al, 2010).…”

Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis

“…Hydroxylamine is a general inhibitor of PLP-dependent enzymes (20). Small chain carboxylic acids (acetate, propionate and butyrate) that resemble alanine also bind to the active site of alanine racemase (21).…”