Prev. Article Table of Contents

Article

Staphylococcus aureus Sortase A Exists as a Dimeric Protein In Vitro^†

Changsheng Lu,^‡ Jie Zhu, Yun Wang, Aiko Umeda, Roshani B. Cowmeadow, Eric Lai, Gabrielle N. Moreno, Maria D. Person, and Zhiwen Zhang*

Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712

Biochemistry, 2007, 46 (32), pp 9346–9354

DOI: 10.1021/bi700519w

Publication Date (Web): July 21, 2007

†

Supported by The Welch Foundation (F1618), American Heart Association (0665198Y), and start-up funds from College of Pharmacy at The University of Texas at Austin.

‡

Current address: Coordination Chemistry Institute, State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, China.

Address correspondence to this author. Tel: 512-471-4551. Fax: 512-232-2606. E-mail: zhang@mail.utexas.edu.

Abstract

We report the first direct observation of the self-association behavior of the Staphylococcus aureus sortase A (SrtA) transpeptidase. Formation of a SrtA dimer was observed under native conditions by polyacrylamide gel electrophoresis and fast protein liquid chromatography (FPLC). Subsequent peptide mass fingerprinting and protein sequencing experiments confirmed the dimeric form of the SrtA protein. Furthermore, SrtA can be selectively cross-linked both in vitro and in Escherichia coli. Multiple samples of enzyme were subjected to analytical sedimentation equilibrium ultracentrifugation to obtain an apparent K_d for dimer formation of about 55 μM. Finally, enzyme kinetic studies suggested that the dimeric form of SrtA is more active than the monomeric enzyme. Discovery of SrtA dimerization may have significant implications for understanding microbial physiology and developing new antibiotics.

View: Full Text HTML | Hi-Res PDF