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.

Three-dimensional structure of a hyperthermophilic 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus.

The structure of 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) has been determined alone, as ternary complexes with sulfate plus substrates 5'-deoxy-5'-methylthioadenosine, adenosine, or guanosine, or with the noncleavable substrate analog Formycin B and as binary complexes with phosphate or sulfate alone. The structure of unliganded SsMTAP was refined at 2.5-A resolution and the structures of the complexes were refined at resolutions ranging from 1.6 to 2.0 A. SsMTAP is unusual both for its broad substrate specificity and for its extreme thermal stability. The hexameric structure of SsMTAP is similar to that of purine-nucleoside phosphorylase (PNP) from Escherichia coli, however, only SsMTAP accepts 5'-deoxy-5'-methylthioadenosine as a substrate. The active site of SsMTAP is similar to that of E. coli PNP with 13 of 18 nearest residues being identical. The main differences are at Thr(89), which corresponds to serine in E. coli PNP, and Glu(163), which corresponds to proline in E. coli PNP. In addition, a water molecule is found near the purine N-7 position in the guanosine complex of SsMTAP. Thr(89) is near the 5'-position of the nucleoside and may account for the ability of SsMTAP to accept either hydrophobic or hydrophilic substituents in that position. Unlike E. coli PNP, the structures of SsMTAP reveal a substrate-induced conformational change involving Glu(163). This residue is located at the interface between subunits and swings in toward the active site upon nucleoside binding. The high-resolution structures of SsMTAP suggest that the transition state is stabilized in different ways for 6-amino versus 6-oxo substrates. SsMTAP has optimal activity at 120 degrees C and retains full activity after 2 h at 100 degrees C. Examination of the three-dimensional structure of SsMTAP suggests that unlike most thermophilic enzymes, disulfide linkages play a key in role in its thermal stability.

Crystal structure of 4-methyl-5-beta-hydroxyethylthiazole kinase from Bacillus subtilis at 1.5 A resolution.

4-Methyl-5-beta-hydroxyethylthiazole kinase (ThiK) catalyzes the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole (Thz). This enzyme is a salvage enzyme in the thiamin biosynthetic pathway and enables the cell to use recycled Thz as an alternative to its synthesis from 1-deoxy-D-xylulose-5-phosphate, cysteine, and tyrosine. The structure of ThiK in the rhombohedral crystal form has been determined to 1.5 A resolution and refined to a final R-factor of 21. 6% (R-free 25.1%). The structures of the enzyme/Thz complex and the enzyme/Thz-phosphate/ATP complex have also been determined. ThiK is a trimer of identical subunits. Each subunit contains a large nine-stranded central beta-sheet flanked by helices. The overall fold is similar to that of ribokinase and adenosine kinase, although sequence similarity is not immediately apparent. The area of greatest similarity occurs in the ATP-binding site where several key residues are highly conserved. Unlike adenosine kinase and ribokinase, in which the active site is located between two domains within a single subunit, the ThiK active site it formed at the interface between two subunits within the trimer. The structure of the enzyme/ATP/Thz-phosphate complex suggests that phosphate transfer occurs by an inline mechanism. Although this mechanism is similar to that proposed for both ribokinase and adenosine kinase, ThiK lacks an absolutely conserved Asp thought to be important for catalysis in the other two enzymes. Instead, ThiK has a conserved cysteine (Cys198) in this position. When this Cys is mutated to Asp, the enzymatic activity increases 10-fold. Further sequence analysis suggests that another thiamin biosynthetic enzyme (ThiD), which catalyzes the formation of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate by two sequential phosphorylation reactions, belongs to the same family of small molecule kinases.

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding.

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gamma phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding.

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gi phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.

Characterization of the active site of ADP-ribosyl cyclase.

ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and that Glu(179) may indeed be the catalytic residue.

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.

Structure of human adenosine kinase at 1.5 A resolution.

Adenosine kinase (AK) is a key enzyme in the regulation of extracellular adenosine and intracellular adenylate levels. Inhibitors of adenosine kinase elevate adenosine to levels that activate nearby adenosine receptors and produce a wide variety of therapeutically beneficial activities. Accordingly, AK is a promising target for new analgesic, neuroprotective, and cardioprotective agents. We determined the structure of human adenosine kinase by X-ray crystallography using MAD phasing techniques and refined the structure to 1.5 A resolution. The enzyme structure consisted of one large alpha/beta domain with nine beta-strands, eight alpha-helices, and one small alpha/beta-domain with five beta-strands and two alpha-helices. The active site is formed along the edge of the beta-sheet in the large domain while the small domain acts as a lid to cover the upper face of the active site. The overall structure is similar to the recently reported structure of ribokinase from Escherichia coli [Sigrell et al. (1998) Structure 6, 183-193]. The structure of ribokinase was determined at 1.8 A resolution and represents the first structure of a new family of carbohydrate kinases. Two molecules of adenosine were present in the AK crystal structure with one adenosine molecule located in a site that matches the ribose site in ribokinase and probably represents the substrate-binding site. The second adenosine site overlaps the ADP site in ribokinase and probably represents the ATP site. A Mg2+ ion binding site is observed in a trough between the two adenosine sites. The structure of the active site is consistent with the observed substrate specificity. The active-site model suggests that Asp300 is an important catalytic residue involved in the deprotonation of the 5'-hydroxyl during the phosphate transfer.