Publications

• The crystal structure of spermidine/spermine N1-acetyltransferase in complex with spermine provides insights into substrate binding and catalysis.

  Montemayor E.J., Hoffman D.W.

  The enzyme spermidine/spermine N (1)-acetyltransferase (SSAT) catalyzes the transfer of acetyl groups from acetylcoenzyme A to spermidine and spermine, as part of a polyamine degradation pathway. This work describes the crystal structure of SSAT in complex with coenzyme A, with and without bound s ... >> More

  The enzyme spermidine/spermine N (1)-acetyltransferase (SSAT) catalyzes the transfer of acetyl groups from acetylcoenzyme A to spermidine and spermine, as part of a polyamine degradation pathway. This work describes the crystal structure of SSAT in complex with coenzyme A, with and without bound spermine. The complex with spermine provides a direct view of substrate binding by an SSAT and demonstrates structural plasticity near the active site of the enzyme. Associated water molecules bridge several of the intermolecular contacts between spermine and the enzyme and form a "proton wire" between the side chain of Glu92 and the N1 amine of spermine. A single water molecule can also be seen forming hydrogen bonds with the side chains of Glu92, Asp93, and the N4 amine of spermine. Site-directed mutation of Glu92 to glutamine had a detrimental effect on both substrate binding and catalysis and shifted the optimal pH for enzyme activity further into alkaline solution conditions, while mutation of Asp93 to asparagine affected both substrate binding and catalysis without changing the pH dependence of the enzyme. Considered together, the structural and kinetic data suggest that Glu92 functions as a catalytic base to drive an otherwise unfavorable deprotonation step at physiological pH. << Less

  Biochemistry 47:9145-9153(2008) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Cryptosporidium parvum spermidine/spermine N1-acetyltransferase exhibits different characteristics from the host enzyme.

  Yarlett N., Wu G., Waters W.R., Harp J.A., Wannemuehler M.J., Morada M., Athanasopoulos D., Martinez M.P., Upton S.J., Marton L.J., Frydman B.J.

  Cryptosporidosis is a severe opportunistic infection of immuno-compromised individuals for which no reliable therapy exists. The parasite scavenges host-derived polyamines, particularly spermine, which is then converted to the lower polyamines by the combined action of spermidine/spermine N(1)-ace ... >> More

  Cryptosporidosis is a severe opportunistic infection of immuno-compromised individuals for which no reliable therapy exists. The parasite scavenges host-derived polyamines, particularly spermine, which is then converted to the lower polyamines by the combined action of spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine oxidase (PAO). We have isolated and expressed the Cryptosporidium parvum SSAT for kinetic and molecular comparison with the host enzyme. The CpSSAT is a homotetramer with a subunit molecular mass of 18 kDa and low sequence similarity to higher eukaryotes but maintains the critical arginine residues in the active site. The CpSSAT had an activity of 299 nmol(-1)min(-1)(mg of protein)(-1) and exhibits an ordered Bi-Bi kinetics with preferred substrate specificity for spermine. Polyamine analogues having unsaturated central carbons were found to exhibit mixed inhibition kinetics of the CpSSAT. The cis-analogues were more effective inhibitors of the CpSSAT with lower K(i) values than the trans-analogues. Experiments aimed at determining the ratio of the time of the analogue in the enzyme active site to that spent out (in-out time: delta ln E/deltat) confirmed the higher efficiency of the cis-analogues as inhibitors of the CpSSAT. The results of this study reveal that the C. parvum SSAT may provide a rational target for drug design. << Less

  Mol. Biochem. Parasitol. 152:170-180(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Occurrence and induction of spermidine-N1-acetyltransferase in Escherichia coli.

  Matsui I., Kamei M., Otani S., Morisawa S., Pegg A.E.

  Biochem. Biophys. Res. Commun. 106:1155-1160(1982) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification and characterization of spermidine/spermine N1-acetyltransferase from rat liver.

  Ragione F.D., Pegg A.E.

  Biochemistry 21:6152-6158(1982) [PubMed] [EuropePMC]

• Properties and structure of spermidine acetyltransferase in Escherichia coli.

  Fukuchi J., Kashiwagi K., Takio K., Igarashi K.

  Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold. The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits. The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine. The Km val ... >> More

  Spermidine acetyltransferase (SAT) from Escherichia coli was purified about 40,000-fold. The molecular mass of native SAT was 95 kDa, and it consisted of four identical subunits. The products formed from the reaction of acetyl-CoA with spermidine by SAT were N1- and N8-acetylspermidine. The Km values for acetyl-CoA, spermidine, and spermine were 2 microM, 1.29 mM, and 220 microM, respectively. The enzymatic activity increased by 2.5-3.5-fold under the condition of poor nutrition but not in response to cold shock or high pH. By using synthetic oliogonucleotides deduced from amino acid sequences of the peptides in SAT, a polymerase chain reaction product with a length of 250 nucleotides was obtained. Using this polymerase chain reaction product, the gene encoding SAT (speG) was cloned and mapped at 35.6 min in the E. coli chromosome. E. coli cells transformed with the cloned speG gene increased SAT activity by 8-40-fold. The gene encoded a 186-amino acid protein, but SAT consisted of 185 amino acids because the initiator methionine was liberated from the protein. Thus, the predicted molecular mass was 21,756 Da. Significant similarity to aminoglycoside acetyltransferase and peptide N-acetyltransferase was observed in the amino acid sequence 87-141, and some similarity with spermidine-preferential binding protein (potD protein) in the spermidine-preferential uptake system was observed in the amino acid sequence 122-141. The results suggest that the active center of SAT may be located in the COOH-terminal portion. << Less

  J. Biol. Chem. 269:22581-22585(1994) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Mechanistic and structural analysis of human spermidine/spermine N1-acetyltransferase.

  Hegde S.S., Chandler J., Vetting M.W., Yu M., Blanchard J.S.

  The N1-acetylation of spermidine and spermine by spermidine/spermine acetyltransferase (SSAT) is a crucial step in the regulation of the cellular polyamine levels in eukaryotic cells. Altered polyamine levels are associated with a variety of cancers as well as other diseases, and key enzymes in th ... >> More

  The N1-acetylation of spermidine and spermine by spermidine/spermine acetyltransferase (SSAT) is a crucial step in the regulation of the cellular polyamine levels in eukaryotic cells. Altered polyamine levels are associated with a variety of cancers as well as other diseases, and key enzymes in the polyamine pathway, including SSAT, are being explored as potential therapeutic drug targets. We have expressed and purified human SSAT in Escherichia coli and characterized its kinetic and chemical mechanism. Initial velocity and inhibition studies support a random sequential mechanism for the enzyme. The bisubstrate analogue, N1-spermine-acetyl-coenzyme A, exhibited linear, competitive inhibition against both substrates with a true Ki of 6 nM. The pH-activity profile was bell-shaped, depending on the ionization state of two groups exhibiting apparent pKa values of 7.27 and 8.87. The three-dimensional crystal structure of SSAT with bound bisubstrate inhibitor was determined at 2.3 A resolution. The structure of the SSAT-spermine-acetyl-coenzyme A complex suggested that Tyr140 acts as general acid and Glu92, through one or more water molecules, acts as the general base during catalysis. On the basis of kinetic properties, pH dependence, and structural information, we propose an acid/base-assisted reaction catalyzed by SSAT, involving a ternary complex. << Less

  Biochemistry 46:7187-7195(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structural and functional evidence for Bacillus subtilis PaiA as a novel N1-spermidine/spermine acetyltransferase.

  Forouhar F., Lee I.-S., Vujcic J., Vujcic S., Shen J., Vorobiev S.M., Xiao R., Acton T.B., Montelione G.T., Porter C.W., Tong L.

  Bacillus subtilis PaiA has been implicated in the negative control of sporulation as well as production of degradative enzymes. PaiA shares recognizable sequence homology with N-acetyltransferases, including those that can acetylate spermidine/spermine substrates. We have determined the crystal st ... >> More

  Bacillus subtilis PaiA has been implicated in the negative control of sporulation as well as production of degradative enzymes. PaiA shares recognizable sequence homology with N-acetyltransferases, including those that can acetylate spermidine/spermine substrates. We have determined the crystal structure of PaiA in complex with CoA at 1.9 A resolution and found that PaiA is a member of the N-acetyltransferase superfamily of enzymes. Unexpectedly, we observed the binding of an oxidized CoA dimer in the active site of PaiA, and the structural information suggests the substrates of the enzyme could be linear, positively charged compounds. Our biochemical characterization is also consistent with this possibility, since purified PaiA possesses N1-acetyltransferase activity toward polyamine substrates including spermidine and spermine. Further, conditional overexpression of PaiA in bacteria results in increased acetylation of endogenous spermidine pools. Thus, our structural and biochemical analyses indicate that PaiA is a novel N-acetyltransferase capable of acetylating both spermidine and spermine. In this way, the pai operon may function in regulating intracellular polyamine concentrations and/or binding capabilities. In addition to preventing toxicity due to polyamine excess, this function may also serve to regulate expression of certain bacterial gene products such as those involved in sporulation. << Less

  J. Biol. Chem. 280:40328-40336(2005) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structures of wild-type and mutant human spermidine/spermine N1-acetyltransferase, a potential therapeutic drug target.

  Bewley M.C., Graziano V., Jiang J., Matz E., Studier F.W., Pegg A.E., Coleman C.S., Flanagan J.M.

  Spermidine/spermine N1-acetyltransferase (SSAT) is a key enzyme in the control of polyamine levels in human cells, as acetylation of spermidine and spermine triggers export or degradation. Increased intracellular polyamine levels accompany several types of cancers as well as other human diseases, ... >> More

  Spermidine/spermine N1-acetyltransferase (SSAT) is a key enzyme in the control of polyamine levels in human cells, as acetylation of spermidine and spermine triggers export or degradation. Increased intracellular polyamine levels accompany several types of cancers as well as other human diseases, and compounds that affect the expression, activity, or stability of SSAT are being explored as potential therapeutic drugs. We have expressed human SSAT from the cloned cDNA in Escherichia coli and have determined high-resolution structures of wild-type and mutant SSAT, as the free dimer and in binary and ternary complexes with CoA, acetyl-CoA (AcCoA), spermine, and the inhibitor N1,N11bis-(ethyl)-norspermine (BE-3-3-3). These structures show details of binding sites for cofactor, substrates, and inhibitor and provide a framework to understand enzymatic activity, mutations, and the action of potential drugs. Two dimer conformations were observed: a symmetric form with two open surface channels capable of binding substrate or cofactor, and an asymmetric form in which only one of the surface channels appears capable of binding and acetylating polyamines. SSAT was found to self-acetylate lysine-26 in the presence of AcCoA and absence of substrate, a reaction apparently catalzyed by AcCoA bound in the second channel of the asymmetric dimer. These unexpected and intriguing complexities seem likely to have some as yet undefined role in regulating SSAT activity or stability as a part of polyamine homeostasis. Sequence signatures group SSAT with proteins that appear to have thialysine Nepsilon-acetyltransferase activity. << Less

  Proc. Natl. Acad. Sci. U.S.A. 103:2063-2068(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.