Human spermidine-spermine N1-acetyltransferase

Target ID:

SAT

Aliases:

DC21KFSDSATSSATSSAT-1

Deposition Date:

Sunday 5th February 2006

Families:

Transferases

Related Diseases:

Metabolism

Structure type:

apo

Download Coordinates:

2FXF

Authors:

J.R.MIN,H.WU,H.ZENG,P.LOPPNAU,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,A.N.PLOTNIKOV,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

2FXF

tabs

Structure Details

The nearly ubiquitous polyamines (putrescine, spermidine and spermine)
are mediators of cell proliferation and differentiation.
They may act as ligands at multiple sites on DNA, RNA, proteins, phospholipids,
and nucleotide triphosphates.
In normal cells, polyamine levels are intricately controlled by biosynthetic and catabolic enzymes.
The strong correlation between polyamine synthesis and cell proliferation
makes the polyamine biosynthetic and catabolic enzymes attractive targets
for the development of anti-proliferative therapeutics.

Spermidine/spermine N1-acetyltransferase (SPD/SPM acetyltransferase, SAT) is a rate-limiting enzyme in the catabolic pathway of polyamine metabolism.
It catalyzes the N1-acetylation of spermidine and spermine and, by the successive activity of polyamine oxidase, spermine can be converted to spermidine and spermidine to putrescine.
SAT is strongly induced by polyamines and polyamine analogues.
This induction is important in protection of the cells against the toxic effect of excess polyamines.
Here we solved the crystal structure of human spermidine/spermine N1-acetyltransferase in complex with acetylcoenzyme A at 2.0 Å resolution.
The structure provides us important information on substrate/co-factor binding site.

Materials and Methods

SAT

PDB:2FXF

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:4506789
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).

Construct

Prelude:
Sequence:
Vector:pET28a-LIC

Growth

Medium:
Antibiotics:
Procedure:SAT was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 µg/mL of kanamycin. Cell were grown at 37oC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15oC.

Purification

Procedure

Extraction

Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80°C. For the purification the cell paste was thawed and resuspended in lysis buffer (1X phosphate-buffered-saline, 0.25 M NaCl, 5 mM imidazole, 2 mM ß-mercaptoethanol, 5% glycerol) with protease inhibitor (0.1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:
Ligand
MassSpec:
Crystallization:Purified SAT was complexed with acetylcoenzyme A (AcCoA, Sigma) at 1:10 molar ratio of protein:AcCoA and crystallized using the hanging drop vapor diffusion method at 20 °C by mixing 1.5 µl of the protein solution with 1.5 µl of the reservoir solution containing 18% PEG4000, 0.2 M Ca Acetate, 0.1 M HEPES, pH 7.5.
NMR Spectroscopy:
Data Collection:
Data Processing: