W0428

The Structure of Human SSAT Implies a Model for Regulating its Turnover: A Tale of Two Lysines. Maria Bewley, Vito Graziano, Jiangsheng Jiang, F. William Studier, John Flanagan, Biology Dept., Brookhaven National Laboratory, Bldg. 463, PO Box 5000, Upton, NY 11973 USA.

Polyamine levels are tightly regulated in human cells, and deficiencies in this regulation are associated with certain human diseases, including Alzheimer’s disease and some cancers. Spermine/Spermidine Acetyltransferase (SSAT) is a key enzyme in the control of polyamine levels, catalyzing a reaction that reduces cellular polyamine levels. The level of SSAT in the cell is regulated both at the level of transcription and by altering the turnover of the enzyme by the ubiquitin/proteasome pathway, thereby rapidly and efficiently matching enzyme level to demand. We have determined the X-ray crystal structure of human SSAT enzyme alone, in complex with its cofactor AcetylCoA (AcCoA), and in a ternary complex with unacetlyated CoA and the substrate spermine (Spm). The protein is a homodimer containing two putative active sites in long channels at the dimer interface. Surprisingly, one subunit in the dimer was acetylated on a single lysine (K26). Lysine-26 is located in a loop involved in substrate binding, and bound substrate was observed only in the active site in which K26 was not acetylated. Substitution of K26 with arginine prevented acetylation and produced an enzyme that bound substrate at both active sites. Structural and biochemical information suggest that auto-acetylation at K26 is part of a novel mechanism for regulating the ubiquitin-dependent degradation of SSAT.