No 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...

S-Adenosylmethionine decarboxylase (AdoMetDC) is synthesized as a proenzyme that cleaves itself in a putrescine-stimulated reaction via an N-->O acyl shift and beta-elimination to produce an active enzyme with a catalytically essential pyruvoyl residue at the new N-terminus. N-->O acyl shifts initiate the self-processing of other proteins such as inteins and amidohydrolases, but their mechanisms in such proteins are not well understood. We have solved the crystal structure of the H243A mutant of AdoMetDC to 1.5 A resolution. The mutant protein is trapped in the ester form, providing clear evidence for the structure of the ester intermediate in the processing of pyruvoyl enzymes. In addition, a putrescine molecule is bound in a charged region within the beta-sandwich, and cross-links the two beta-sheets through hydrogen bonds to several acidic residues and ordered water molecules. The high-resolution structure provides insight into the mechanism for the self-processing reaction and provides evidence for the mechanism for simulation of the self-processing reaction by putrescine. Studies of the effects of putrescine or 4-aminobutanol on the processing of mutant AdoMetDC proenzymes are consistent with a model in which a single activator molecule interacts with buried Asp174, Glu178, and Glu256, leading to an alteration in the position of Glu11, resulting in stimulation of self-processing.