Abstract
Haloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Using a representative set of 80 phosphorylated substrates, we characterized the substrate specificities of 23 soluble HADs encoded in the Escherichia coli genome. We identified small molecule phosphatase activity in 21 HADs and beta-phosphoglucomutase activity in one protein. The E. coli HAD phosphatases show high catalytic efficiency and affinity to a wide range of phosphorylated metabolites that are intermediates of various metabolic reactions. Rather than following the classical "one enzyme-one substrate" model, most of the E. coli HADs show remarkably broad and overlapping substrate spectra. At least 12 reactions catalyzed by HADs currently have no EC numbers assigned in Enzyme Nomenclature. Surprisingly, most HADs hydrolyzed small phosphodonors (acetyl phosphate, carbamoyl phosphate, and phosphoramidate), which also serve as substrates for autophosphorylation of the receiver domains of the two-component signal transduction systems. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, YniC. Many of the secondary activities of HADs might have no immediate physiological function but could comprise a reservoir for evolution of novel phosphatases.
Highlights
Haloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, -phosphoglucomutase, phosphonatase, and dehalogenase activities
The Enzymatic Activities of the E. coli HADs—HADs share relatively little overall sequence similarity (ϳ15 to 30% identity), but they can be identified by the presence of three short conserved sequence motifs [3]. 22 soluble HADs encoded by the E. coli genome were purified to homogeneity and screened for phosphatase, dehalogenase, phosphonatase, and -phosphoglucomutase activities. 16 previously uncharacterized HADs showed detectable activity with p-nitrophenyl phosphate (pNPP), a general phosphatase substrate [18] (Table 1)
In the KEGG data base, this periplasmic phosphatase is annotated as an enzyme responsible for intracellular dephosphorylation of ␣-glucose-1-P, our results indicate that both YihX (HAD4) and YidA (HAD13) have at least 2 times lower Km to this substrate, and these enzymes are likely to be the principal phosphatases involved in the intracellular metabolism of ␣-glucose-1-P in E. coli
Summary
Gene Cloning and Protein Purification—For most HADs analyzed in this work, the genes were amplified by PCR from the E. coli DH5␣ genomic DNA and cloned into a modified pET15b (Novagen) as previously described [17]. The production of fructose in enzymatic reactions was determined using an enzyme-coupled assay with fructose dehydrogenase (F5152; Sigma), essentially as previously described [20]. This assay was adapted for 96-well microplates (200-l reaction mixtures). Phosphonatase activity was assayed using phosphonoacetate as a substrate by measuring the release of inorganic phosphate using the Malachite Green reagent as previously described [23]. The substrate profiles across proteins are clustered into groups using the average linkage method (R Foundation for Statistical Computing; available on the World Wide Web at www.R-project.org). Sequence-based phylogenetic trees were constructed using the following methods: (i) neighbor-joining method, as implemented in the NEIGHBOR program of the PHYLIP package [28], (ii) least squares method as implemented in the FITCH program of the PHYLIP package [28], (iii) local maximum likelihood optimization of the least squares tree using the ProtML program of the MOLPHY package [30], (iv) quartet puzzling as implemented in the TREE-PUZZLE program [31], and (v) Markov chain Monte Carlo Bayesian estimation using the MrBayes program [32, 33]
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