Genome-wide Analysis of Substrate Specificities of the Escherichia coli Haloacid Dehalogenase-like Phosphatase Family

Abstract

Most enzymes form families of paralogs whose members are related by sequence and catalyze similar reactions but have evolved specific biological functions. Comprehensive determination of the substrate specificities and selectivities of all metabolic enzymes in an organism is an essential step toward understanding the relationship between the proteome and the metabolome. By the most recent estimate, Escherichia coli possesses at least 1186 metabolic enzymes and 1005 metabolites (1). The most common functional group in the metabolome is phosphate; 35-40% of the metabolites contain a phosphate group (2). The pool of phosphorylated metabolites is controlled by the activity of diverse kinases and phosphatases, of which there are hundreds in the E. coli genome.

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae

“…The targeted peptide sequences are present in the S. cerevisiae MAPKs HOG1, FUS3, and KSS1; in the regulatory proteins REG1 and MMF1; and in subunits of several complexes (Pat1p, RIF1, and NMD2). Although the presence of protein phosphatase activity has not yet been reported for E. coli HADs (25,40), this activity has been proposed for several microbial effector proteins with HAD domains from the pathogenic bacteria Porphyromonas gingivalis and Coxiella burnetii (76,77).…”

“…Gene Cloning, Protein Purification, and Mutagenesis-The genes encoding 15 selected yeast HADs (Table 1) were amplified by PCR from S. cerevisiae genomic DNA and cloned into a modified pET15b vector (Novagen) as described previously (40). Purification of proteins for screening and biochemical characterization was performed as described previously (48).…”

“…Enzymatic Screens and Assays-Purified yeast HADs were initially screened for the presence of phosphatase activity against the general phosphatase substrates pNPP and acetyl phosphate as described previously (40). Secondary phosphatase screens with 93 phosphorylated metabolites (supplemental Table 2) were then performed to identify the preferred in vitro substrates for these proteins (40).…”

“…Secondary phosphatase screens with 93 phosphorylated metabolites (supplemental Table 2) were then performed to identify the preferred in vitro substrates for these proteins (40). Phosphatase activity against phosphorylated metabolites and phosphopeptides was measured spectrophotometrically using the malachite green reagent or a mild phosphate detection method (48).…”

“…In particular, phosphoprotein phosphatase activity has been demonstrated for several eukaryotic HAD-like hydrolases: human CTDP1 (or FCP1; Ser(P) phosphatase) (34), Drosophila and mammalian Eyes absent (Tyr(P) phosphatase) (35)(36)(37), and mammalian chronophin (Ser(P) phosphatase) (38,39). In a previous study, we have experimentally characterized the substrate specificities of the 19 soluble HAD-like phosphatases from E. coli and demonstrated that most of the E. coli HADs show remarkably broad and overlapping substrate profiles, being active against phosphorylated carbohydrates, nucleotides, organic acids, and coenzymes (40). A phylogenetic analysis of the E. coli HADs suggested that their secondary activities might have no direct physiological function, but they could comprise a reservoir for the evolution of phosphatases with novel specificities.…”