The HAD superfamily is a large enzyme family (~19,000 nonredundant sequences) [1] of phosphotransferases (phosphomutases, ATPases and phosphatases) represented in all three kingdoms of life [2-4], and, within each cell, by a large number of homologs (28 in E. coli;35 in Salmonella typhimurium;31 in Pseudomonas aeruginosa;30 in Mycobacterium tuberculosis;31 in Bacillus cereus;24 in Bacteroides fragilis; 24 in Streptococcus pneumoniae;45 in Saccharomyces cerevisiae;84 in Caenorhabditis elegans;169 in Arabidopsis thaliana;292 in Selaginella moeltendorffii;183 in human). As many as 80-90% of the members are phosphatases [5], the vast majority of which have unknown functions. Approximately 40% of the bacterial metabolome is comprised of phosphorylated metabolites [6]. Phosphate substituents are common because they enhance the water solubility of the metabolite as well as its ability to bind to metabolic enzymes with high affinity and specificity. The removal of phosphate groups from phosphorylated metabolites is performed by phosphatases. The "function" of a particular phosphatase is defined by the phosphorylated metabolite that it targets in the cell, i.e., by its "physiological substrate". Thus, the HAD phosphatases meet the demands of cellular processes and metabolic pathways that involve phosphorylated macromolecules and metabolites. Divergence in HAD phosphatase funcfion is based on the divergence of the substrate-recognition elements. The substrate-recognition elements are separate from the catalytic scaffold, which is located in the core domain (Figure 1A). The four pepfide segments or "motifs" which form the active site position the consen/ed Asp nucleophile, Asp acid/base, the Lys/Arg and Ser/Thr phosphate-binding residues and the Mg^* cofactor Asp/Glu binding residues (Figure IB). These residues, in combinafion with the scaffold main-chain elements, form a steric and electrostafic mold that stabilizes the trigonal bipyramidal transifion states/intermediates produced along the reaction pathway (Figure 1B) [7]. The HAD phosphatase substrate recognition elements are located in either a cap domain (as in HAD classes Cl and C2, also known as Type I and Type 11) tethered to the core domain by a solvated linker, or in short loop/helical segments that extend from the core domain (as in the "capless" HAD class CO also known as Type III) (Figure 1A) [8]. Although HAD phosphatases possess the same catalytic site and proceed through the same second partial reaction, they are able to use the specific structural requirements of the substrate-binding step and the subsequent addition-eliminafion steps of the first partial reacfion to discriminate between the physiological substrate and other phosphorylated species (macromolecules and metabolites). The induced fit model, wherein substrate binding is followed by cap domain or loop closure, applies to most HAD phosphatases. Favorable electrostafic interaction between the substrate leaving group and the cap domain/gafing loops will contribute to the substrate-binding affinity. For efficient turnover, the phosphoryl group must be bound in the correct orientation within the catalyfic site. If the substrate-leaving group is too large or too small, nonproductive binding is likely to occur. Thus, the size, shape and electrostafic surface ofthe acfive site region that extends from the catalytic site to the active site entrance can provide significant insight into the identity of the physiological substrate. This serves as the basis for the use of virtual screening (made possible by the Structure Core and Computation Core) to identify candidates for the physiological substrate herein. Substrate specificities defined by experimental activity screens suggest that the typical HAD phosphatase has loose substrate specificity coupled with modest catalytic efficiency. Thus, acfivity screens alone often cannot idenfify the actual physiological substrate. Rather, they provide candidates that can be further interrogated using the tools provided by the Sequence/Genome Analysis Core and Microbiology Core.

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Grabowski, Marek; Niedzialkowska, Ewa; Zimmerman, Matthew D et al. (2016) The impact of structural genomics: the first quindecennial. J Struct Funct Genomics 17:1-16
Zhang, Xinshuai; Carter, Michael S; Vetting, Matthew W et al. (2016) Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars. Proc Natl Acad Sci U S A 113:E4161-9
Pan, Jian-Jung; Ramamoorthy, Gurusankar; Poulter, C Dale (2016) Absolute Configuration of Hydroxysqualene. An Intermediate in Bacterial Hopanoid Biosynthesis. Org Lett 18:512-5
Machovina, Melodie M; Usselman, Robert J; DuBois, Jennifer L (2016) Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations. J Biol Chem 291:17816-28
Yadava, Umesh; Vetting, Matthew W; Al Obaidi, Nawar et al. (2016) Structure of an ABC transporter solute-binding protein specific for the amino sugars glucosamine and galactosamine. Acta Crystallogr F Struct Biol Commun 72:467-72
Vetting, Matthew W; Bouvier, Jason T; Gerlt, John A et al. (2016) Purification, crystallization and structural elucidation of D-galactaro-1,4-lactone cycloisomerase from Agrobacterium tumefaciens involved in pectin degradation. Acta Crystallogr F Struct Biol Commun 72:36-41
Kim, Jungwook; Xiao, Hui; Koh, Junseock et al. (2015) Determinants of the CmoB carboxymethyl transferase utilized for selective tRNA wobble modification. Nucleic Acids Res 43:4602-13
London, Nir; Farelli, Jeremiah D; Brown, Shoshana D et al. (2015) Covalent docking predicts substrates for haloalkanoate dehalogenase superfamily phosphatases. Biochemistry 54:528-37
Wichelecki, Daniel J; Vetting, Matthew W; Chou, Liyushang et al. (2015) ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58. J Biol Chem 290:28963-76
Berman, Helen M; Gabanyi, Margaret J; Groom, Colin R et al. (2015) Data to knowledge: how to get meaning from your result. IUCrJ 2:45-58

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