Crystal structure and biochemical analysis of the dimeric transaminase DoeD provides insights into ectoine degradation.

Abstract

The pyridoxal-5'-phosphate-dependent enzyme DoeD is a L-2,4-diaminobutyric acid (DABA) transaminase that is part of the degradation pathway of the compatible solute ectoine. Ectoines are used by halophilic organisms to maintain osmotic balance under fluctuating salt concentrations (osmoadaptation). Classified under class III ω-aminotransferases, DoeD utilizes substrates with terminal amines, facilitated by dual substrate recognition involving two binding pockets, the O-pocket and the P-pocket. In this study, we have determined the first crystal structure of DoeD at 1.5 Å and conducted a biochemical and biophysical characterization of the dimeric DABA transaminase from the halophilic bacterium and model organism Chromohalobacter salexigens DSM 3043. Our findings reveal that pyruvate is the preferred co-substrate and that DoeD has a broad pH tolerance, minimal salt requirements, and can utilize a variety of amino donors. The crystal structure and substrate specificity studies of this highly expressed and stable DoeD suggest opportunities for enhancing enzymatic activity through targeted mutagenesis, optimizing it for industrial applications in green chemistry for chiral amine synthesis.