Structures of wild-type and mutant human spermidine/spermine N ¹ -acetyltransferase, a potential therapeutic drug target

Abstract

Spermidine͞spermine N 1 -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 highresolution 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 N 1 ,N 11 -bis-(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 Nacetyltransferase activity.GCN5-related N-acetyltransferase family ͉ polyamines ͉ symmetric and asymmetric dimer ͉ self-acetylation ͉ thialysine N -acetyltransferase

Regulation of Ornithine Decarboxylase

“…The protein has a novel arrangement of 8 ␤-strands and two ␣-helices but, very interestingly, has a fold similar to some acetyltransferases including spermidine/spermine N 1 -acetyltransferase (SSAT) (26). Antizyme and SSAT are the two major factors regulating polyamine homeostasis (2,3).…”

Suppression of Exogenous Gene Expression by Spermidine/Spermine N1-Acetyltransferase 1 (SSAT1) Cotransfection

“…SSAT1 Activity Is Required for Suppression of GFP or GFPeIF5A Expression-To determine whether the acetylation activity of SSAT1 is required for suppression of the reporter protein expression, we first compared the effects of the two SSAT1 mutants R101A and R101K with the wild type enzyme. From the crystal structure (24) and earlier mutagenesis study (30,31), Arg 101 is known to be important for the binding of acetyl-CoA. Substitution of this residue with Ala (or Lys) caused loss of activity to less than 5% of the wild type enzyme.…”

“…2, A and B), indicating that the acetylation activity of SSAT1 is crucial for the SSAT1 effects. We further tested additional mutant enzymes, with mutation at the catalytic site (Y140A and Y140F) (24), and those with high K m values for spermidine (30), E152K, R155A, H26A, L56F (32), L56A, and E152K/R155A. None of these SSAT1 mutant enzymes effectively blocked the expression of GFP (data not shown), indicating that the SSAT1 activity is required for the effect.…”

Spermidine/spermine-N1-acetyltransferase: a key metabolic regulator

“…Bottom: the molecular surface of SSAT (left) and TLAT (right) is shown, colored according to element (nitrogen, blue; oxygen, red; carbon, gray; sulfur, yellow), demonstrating the differences between these proteins in surface properties. tion, was the observation that SSAT can self-acetylate at Lys 26 (15). Analysis of the recombinant human protein produced at high concentrations in E. coli showed that half of the monomers had an N-acetyl group on this residue.…”