Crystallization of Liganded Phosphatases in the HAD Superfamily

Abstract Phosphotransferases catalyze reactions on chemically diverse molecules in organisms from all domains of life. The haloalkanoate dehalogenase superfamily (HADSF) is a model system for phosphoryl transfer enzymes as members catalyze phosphoester hydrolase, phosphonate hydrolase, and phosphomutase reactions on sugars, lipids, nucleotides, and peptides. Because these reactions are fundamental to essential metabolic transformations, understanding the mechanism and determinants of substrate specificity in the HADSF is critical. Structure/function relationships in the superfamily have also been leveraged in the development of methodologies for the assignment of enzyme function. Enzyme complexes with substrate, product, and analogs of the ground state or intermediate/transition state can be studied via high-resolution macromolecular crystallography to provide insight to the relative location of residues and ligands, as well as associated enzyme conformational states. This knowledge can aid in inhibitor design for phosphohydrolase reactions and target-specific therapeutics. Here we describe experimental approaches to capture liganded X-ray crystallographic structures of HADSF members. A number of these methods can be employed generally, including other families of phosphohydrolases and enzymes catalyzing phosphoryl transfer.