The structural basis for substrate specificity and inhibition of human S-adenosylmethionine decarboxylase.

S-Adenosylmethionine decarboxylase belongs to a small class of amino acid decarboxylases that use a covalently bound pyruvate as a prosthetic group. It is an essential enzyme for polyamine biosynthesis and provides an important target for the design of anti-parasitic and cancer chemotherapeutic agents. We have determined the structures of S-adenosylmethionine decarboxylase complexed with the competitive inhibitors methylglyoxal bis(guanylhydrazone) and 4-amidinoindan-1-one-2'-amidinohydrazone as well as the irreversible inhibitors 5'-deoxy-5'-[N-methyl-N-[(2-aminooxy)ethyl]amino]adenosine, 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)amino]adenosine, and the methyl ester analogue of S-adenosylmethionine. These structures elucidate residues important for substrate binding and show how those residues interact with both covalently and noncovalently bound inhibitors. S-Adenosylmethionine decarboxylase has a four-layer alphabeta betaalpha sandwich fold with residues from both beta-sheets contributing to substrate and inhibitor binding. The side chains of conserved residues Phe7, Phe223, and Glu247 and the backbone carbonyl of Leu65 play important roles in binding and positioning the ligands. The catalytically important residues Cys82, Ser229, and His243 are positioned near the methionyl group of the substrate. One molecule of putrescine per monomer is observed between the two beta-sheets but far away from the active site. The activating effects of putrescine may be due to conformational changes in the enzyme, to electrostatic effects, or both. The adenosyl moiety of the bound ligand is observed in the unusual syn conformation. The five structures reported here provide a framework for interpretation of S-adenosylmethionine decarboxylase inhibition data and suggest strategies for the development of more potent and more specific inhibitors of S-adenosylmethionine decarboxylase.

Structure of a human S-adenosylmethionine decarboxylase self-processing ester intermediate and mechanism of putrescine stimulation of processing as revealed by the H243A mutant.

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.

Human liver glycogen phosphorylase inhibitors bind at a new allosteric site.

BACKGROUND: Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate for glycolysis. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. RESULTS: The binding site in human liver glycogen phosphorylase (HLGP) for a class of promising antidiabetic agents was identified crystallographically. The site is novel and functions allosterically by stabilizing the inactive conformation of HLGP. The initial view of the complex revealed key structural information and inspired the design of a new class of inhibitors which bind with nanomolar affinity and whose crystal structure is also described. CONCLUSIONS: We have identified the binding site of a new class of allosteric HLGP inhibitors. The crystal structure revealed the details of inhibitor binding, led to the design of a new class of compounds, and should accelerate efforts to develop therapeutically relevant molecules for the treatment of diabetes.

The crystal structure of human S-adenosylmethionine decarboxylase at 2.25 A resolution reveals a novel fold.

BACKGROUND: S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine synthetic pathway, and a well-studied drug target. The AdoMetDC decarboxylation reaction depends upon a pyruvoyl cofactor generated via an intramolecular proenzyme self-cleavage reaction. Both the proenzyme-processing and substrate-decarboxylation reactions are allosterically enhanced by putrescine. Structural elucidation of this enzyme is necessary to fully interpret the existing mutational and inhibitor-binding data, and to suggest further experimental studies. RESULTS: The structure of human AdoMetDC has been determined to 2.25 A resolution using multiwavelength anomalous diffraction (MAD) phasing methods based on 22 selenium-atom positions. The quaternary structure of the mature AdoMetDC is an (alpha beta)2 dimer, where alpha and beta represent the products of the proenzyme self-cleavage reaction. The architecture of each (alpha beta) monomer is a novel four-layer alpha/beta-sandwich fold, comprised of two antiparallel eight-stranded beta sheets flanked by several alpha and 3(10) helices. CONCLUSIONS: The structure and topology of AdoMetDC display internal symmetry, suggesting that this protein may be the product of an ancient gene duplication. The positions of conserved, functionally important residues suggest the location of the active site and a possible binding site for the effector molecule putrescine.