Processing of Mammalian and PlantS-Adenosylmethionine Decarboxylase Proenzymes

Abstract

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl enzyme, and the pyruvate is formed in an intramolecular reaction that cleaves a proenzyme precursor and converts a serine residue into pyruvate. The wild type potato AdoMetDC proenzyme processed much faster than the human proenzyme and did not require putrescine for an optimal rate of processing despite the presence of three acidic residues (equivalent to ) is not present in the potato sequence. The site of potato AdoMetDC proenzyme processing was found to be Ser 73 in the conserved sequence, YVLSESS, which is the equivalent of Ser 68 in the human sequence. Replacement of the serine precursor with threonine or cysteine by site-directed mutagenesis in either the potato or the human AdoMetDC proenzyme did not prevent processing but caused a significant reduction in the rate. Although the COOH-terminal regions of the known eukaryotic AdoMetDCs are not conserved, only relatively small truncations of 8 residues from the human protein and 25 residues from the potato proenzyme were compatible with processing. The maximally truncated proteins show no similarity in COOHterminal amino acid sequence but each contained 46 amino acid residues after the last conserved sequence, suggesting that the length of this section of the protein is essential for maintaining the proenzyme conformation needed for autocatalytic processing. AdoMetDC1 is an essential enzyme for the biosynthesis of polyamines and is one of a small class of decarboxylases that uses a covalently bound pyruvate as a prosthetic group (1, 2). These pyruvoyl-dependent decarboxylases form amines such as histamine, decarboxylated S-adenosylmethionine, phosphatidylethanolamine (a component of membrane phospholipids), and ␤-alanine (a precursor of coenzyme A), which are all of critical importance in cellular physiology and provide an important target for drug design. The mechanism of formation of the prosthetic group has been studied extensively using histidine decarboxylase from Lactobacillus (1, 3-6), and more preliminary studies with other decarboxylases including AdoMetDC (7-9) suggest that the mechanism is similar (Fig. 1). In all cases, the enzyme is synthesized as a proenzyme that then undergoes an intramolecular cleavage reaction forming the two subunits and generating the pyruvate at the amino terminus of the ␣ subunit from a serine precursor residue. Cleavage takes place via the formation of an intermediate ester resulting from a nucleophilic attack of this serine residue at the amide carbonyl group of the preceding amino acid. This is followed by ␤-elimination to form the ␤ subunit and the ␣ subunit containing a dehydroalanine at its amino terminus. The dehydroalanine then loses ammonia and is converted to pyruvate via the formation of imine and carbinolamine intermediates (1-3). The initial rearrangement step of this reaction to form a peptide ester linked to the hydroxyl side chain of serine is identical to that involved in protein splicing reactions (10, 11). Further information on such cleavage...

Cloning and expression of the S-adenosylmethionine decarboxylase gene of Neurospora crassa and processing of its product

“…The amino acid residues corresponding to those of the human proenzyme previously shown to be required for catalytic activity (Glu8, Glu11, Ser68, Cys82, Ser229, and His243 in the human sequence), and putrescine stimulation of processing and activity (Glu11, Glu15, Arg76, Lys80, Asp174, Glu178, and Glu256) are identical or similar in the N. crassa AdoMetDC (Fig. 3) (Stanley and Pegg 1991;Stanley et al 1994;Xiong et al 1997Xiong et al , 1999 . The conservation of these residues suggests that proenzyme processing and the catalytic activity of the N. crassa enzyme might be stimulated by putrescine.…”

“…By 30 min after inhibition of translation, virtually all the proenzyme was processed, with a corresponding increase in the intensity of the band representing the mature a subunit. This is rapid compared to the human AdoMetDC: only 15% of this precursor is converted to the mature enzyme after 30 min in the absence of putrescine (Xiong et al 1997). A previous study (Xiong et al 1997) found that the TNT reticulocyte lysate, used in these experiments, contained insucient putrescine (<1 nM) to stimulate the processing of human AdoMetDC.…”

“…This is rapid compared to the human AdoMetDC: only 15% of this precursor is converted to the mature enzyme after 30 min in the absence of putrescine (Xiong et al 1997). A previous study (Xiong et al 1997) found that the TNT reticulocyte lysate, used in these experiments, contained insucient putrescine (<1 nM) to stimulate the processing of human AdoMetDC. Therefore, the rapid processing of the N. crassa proenzyme is unlikely to be caused by putrescine in the Fig.…”

“…The pSP6-SPE2 plasmid, in which the coding region for the N. crassa AdoMetDC proenzyme lies downstream from the SP6 promoter, was used in transcription and translation reactions with the TNT SP6 Coupled Reticulocyte Lysate system (Promega) to measure AdoMetDC processing by the method of Xiong et al (1997), who established that this preparation contains less than 1 nM putrescine. Reactions were carried out following the manufacturer's protocol, with the modi®cations indicated below.…”

Structural Roles of Cysteine 50 and Cysteine 230 Residues in Arabidopsis thaliana S-Adenosylmethionine Decarboxylase

“…All of the known sequences, except that from Escherichia coli, show a significant degree of similarity with about 50 fully conserved residues in the core region of 300 amino acids. This includes the sequences (KTCGTT) that contain an essential active site lysine and cysteine residues (Stanley and Pegg, 1991;Stanley et al, 1994;Xiong et al, 1997). In spite of rather extensive studies on the functionally important residues of AdoMetDC, almost all of the studies were confined to the enzyme active site (Stanley and Pegg, 1991;Stanley et al, 1994;Xiong et al, 1999), except the essential Cys 83 residue in the active site.…”