Structure of a Human S-Adenosylmethionine Decarboxylase Self-Processing Ester Intermediate and Mechanism of Putrescine Stimulation of Processing As Revealed by the H243A Mutant^,

Abstract

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.

Catalytic Properties of the Archaeal S-Adenosylmethionine Decarboxylase from Methanococcus jannaschii

“…Mass spectrometry of proteins modified by the latter pathway revealed adducts with mass increases of ϩ57 and ϩ75 daltons resulting from the addition of CH 2 -CH 2 -CHO and the hydrate thereof (15). In contrast the mass spectrum of the M. jannaschii decarboxylase as isolated from E. coli showed that this protein is not detectably modified in vivo when expressed either at low levels (24) or at the ϳ40-fold greater levels of expression obtained in the present work (Fig.…”

“…Bennett et al (13)). The human and potato enzymes have been characterized by crystallographic and functional studies, which have revealed much about the protein structure and active site, as well as the allosteric nature of putrescine activation (13)(14)(15)(16)(17)(18). A second class of AdoMet decarboxylase with a distinct sequence is typified by the Mg 2ϩ -dependent Escherichia coli enzyme that is composed of 12.4-and 18-kDa subunits in an (␣␤) 4 arrangement (7, 15, 19 -23).…”

Allosteric regulation of an essential trypanosome polyamine biosynthetic enzyme by a catalytically dead homolog

“…2A). However, the prozyme is missing several critical residues required for processing and catalytic activity (10,14,15,17,20). Therefore the prozyme is unlikely to display AdoMetDC activity and must have a unique function.…”

“…Human AdoMetDC is a homodimer, and both the processing reaction and decarboxylation of AdoMet are stimulated by putrescine (10,16). The x-ray structure shows that the active sites sit in a large cleft between ␤-sheets distal from the dimer interface and that the putrescine-binding sites are formed by a group of acidic residues in the ␤-sandwich core Ϸ15 Å from the active sites (14,17). This site is eliminated in the structure of the monomeric plant enzyme, which is fully active without putrescine (18).…”

“…The pyruvoyl-moiety derives from an autocatalytic cleavage reaction that generates the active enzyme consisting of two chains (␤␣), with the pyruvoyl group formed at the N terminus of the ␣-chain (14,15). Human AdoMetDC is a homodimer, and both the processing reaction and decarboxylation of AdoMet are stimulated by putrescine (10,16).…”