Structural and Functional Evidence for Bacillus subtilis PaiA as a Novel N1-Spermidine/Spermine Acetyltransferase

Farhad Forouhar,In-Sun Lee,Jelena Vujcic,Slavoljub Vujcic,Jianwei Shen,Sergey M Vorobiev,Rong Xiao,Thomas B Acton,Gaetano T Montelione,Carl W Porter,Liang Tong

doi:10.1074/jbc.m505332200

Abstract

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.

Highlights

Nized based on the amino acid sequence of the protein PaiA, and it was suggested that PaiA may be a DNA-binding protein [1]
In B. subtilis, the BltD gene product was found to be a membrane-bound spermidine/spermine N1-acetyltransferase (SSAT),2 which participates in the export of polyamines [6, 7]
The structure confirms that PaiA is a member of the N-acetyltransferase superfamily

Summary

MATERIALS AND METHODS

Expression and Purification of PaiA—The production of PaiA protein was carried out as part of the high throughput protein production process of the Northeast Structural Genomics Consortium [8]. Fractions containing partially purified PaiA were pooled, and buffer conditions providing monomeric samples were optimized by analytical gel filtration detected by static light scattering, as described elsewhere [8]. The purified PaiA protein was concentrated to 10 mg/ml, flash-frozen in aliquots, and used for crystallization screening. The reaction mixture (final volume 50 ␮l) included 10 ␮l of 0.5 M Tris buffer, 5 ␮l of 30 mM substrate (final concentration, 3.0 mM), 10 ␮l of double distilled H2O, 5 ␮l of 1 mM [14C]acetyl-CoA (60 mCi/mmol; PerkinElmer Life Sciences; final concentration, 100 ␮M) and 20 ␮l containing 125 ng of purified PaiA protein. A detailed kinetic analysis was performed by varying the concentration of potential polyamine substrates from 10 ␮M to 3.0 mM in the presence of saturating levels (100 ␮M) of acetyl-CoA [17]. The assay detects nonacetylated and acetylated polyamines, including N1-acetylspermidine, N 8-acetylspermidine, and N1,N12-diacetylspermine

RESULTS AND DISCUSSION

Resolution range used in refinement

Polyamine poolsb