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    STUDIA BIOLOGIA - Issue no. 1 / 2019  
         
  Article:   MAKING THE STRESS-PROTECTANT ECTOINE: STRUCTURAL AND BIOCHEMICAL INSIGHTS INTO THE ACETYLTRANSFERASE EctA.

Authors:  ALEXANDRA A. RICHTER, STEFANIE KOBUS, LAURA CZECH, ASTRID HÖPPNER, JAN ZARZYCKI, TOBIAS ERB, LUKAS LAUTERBACH, JEROEN S. DICKSCHAT, ERHARD BREMER, SANDER H.J. SMITS. 		 
       
         
  Abstract:   Ectoine is a compatible solute widely synthesized by microorganisms as an osmostress protectant. Due to its stabilizing effect on macromolecules, it is adressed in the literature as a chemical chaperone and used for medical and biotechnological applications. The 2,4-diaminobutyrate acetyltransferase (EctA) catalyzes the second step of the ectoine synthesis pathway, the regio-specific acetylation of 2,4-diaminobutyrate (DAB) to N-γ-acetyl-2,4-diaminobutyric acid (ADABA). Here we focus on the biochemical and structural characteristics of EctA from the thermo-tolerant bacterium Paenibacillus lautus. A codon-optimized ectA gene of P. lautus was cloned into expression vectors for heterologous expression in Escherichia coli. The produced protein was purified, used for enzymatic assays, and crystallized. The enzyme activity of EctA was assessed by photometry-based assays measuring the formation of the co-product CoA. Biochemical characterization of the wildtype enzyme showed optimal activity conditions at high temperature (>40 °C) and high pH-values (8.5-9.5). Crystal structures of apo-EctA, and EctA in complex with the substrate DAB and CoA were obtained. These revealed a functional dimer and illuminated the architecture of the enzymes active site. Putative DAB-binding amino acids were verified by site-directed mutagenesis studies.

Keywords: acetyltransferase, compatible solute, crystal structure, ectoine, osmostress protection. 		 
         
     
         
         
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