Deciphering How Protonation State Supports Catalysis in Bacterial KDN9P Phosphatase

KAREN ALLEN; TYREL BRIAN; JAVIER MARCELO GONZALEZ; JOHN PAUL BACIK; CLIFF UNKEFER; TOBIAS SCHRADER; DEBRA DUNNAWAY-MARIANO; ZOE S. FISHER

Albuquerque

Congreso; American Crystallographic Association Annual Meeting 2014; 2014

American Crystallographic Association

The haloalkanoate dehalogenase superfamily (HADSF) of enzymes is a ubiquitoussuperfamily represented in the proteomes of organisms from all three domains of life,wherein its members participate in numerous diverse biological processes. Neutrondiffraction was undertaken to determine the protonation states of residues that form theconserved catalytic motif of HADSF phosphatases. Knowledge of the protonation states ofactive-site residues in the will facilitate the understanding of catalysis and further thedevelopment of superior docking protocols for structure-based substrate ("function")prediction in the enzyme family. We have determined a 2.3 Å neutron structure of2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9PP) from Bacteriodesthetaiotaomicron co-refined with a 1.8 Å X-ray structure (both at pH 8.5) which clearlyshow that the unliganded enzyme in 2Fo-Fc nuclear density maps that the Asp nucleophileis unprotonated and that the Asp general/acid base catalyst is in a salt bridge with a lysineammonium group wherein the carboxylate bears no nuclear density for the proton. Theresult supports a model for catalysis which can be extrapolated to the otherphosphohydrolases in the superfamily and which is consistent with ligand-inducedconformational positioning of the catalytic residues. This work is supported by NationalInstitute of Health U54 GM093342 (to KNA and DD-M).