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Understanding the functions of every protein in the proteome is one of the great challenges of the postgenomic era. Global genome sequencing efforts revealed that in any genome 30-50% of genes encode proteins with unknown function (hypothetical proteins). To directly test purified hypothetical proteins for catalytic activity, the authors have designed a series of general and specific enzymatic screens. The described screens are designed to detect hydrolases (phosphatases, phosphodiesterases, proteases, and esterases), and oxidoreductases (dehydrogenases and oxidases). The general screens use either general chromogenic substrates or pools of substrates. The positive hits with the model substrates are then tested in the secondary screens with a set of potential natural substrates, or the substrate pools can be deconvoluted to identify the preferred in vitro substrate. The identification of a biochemical activity of a hypothetical protein helps to determine its cellular role.

In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with "known" function are mis-annotated. Several global approaches are routinely employed to predict function, including sophisticated sequence analysis, gene expression, protein interaction, and protein structure. In the first coupling of genomics and enzymology, Phizicky and colleagues undertook a screen for specific enzymes using large pools of partially purified proteins and specific enzymatic assays. Here we present an overview of the further developments of this approach, which involve the use of general enzymatic assays to screen individually purified proteins for enzymatic activity. The assays have relaxed substrate specificity and are designed to identify the subclass or sub-subclasses of enzymes (phosphatase, phosphodiesterase/nuclease, protease, esterase, dehydrogenase, and oxidase) to which the unknown protein belongs. Further biochemical characterization of proteins can be facilitated by the application of secondary screens with natural substrates (substrate profiling). We demonstrate here the feasibility and merits of this approach for hydrolases and oxidoreductases, two very broad and important classes of enzymes. Application of general enzymatic screens and substrate profiling can greatly speed up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches.

The Francisella speciesencode 4 main acid phosphatases (Acp) that are potentially involved in pathogenesis through currently unknown mechanisms. Only 2 of these enzymes, AcpA and AcpC, have been biochemically characterized to date. In this work we describe the catalytic properties of Francisella tularensis AcpB utilizing an array of 120 phosphorylated substrates. In contrast to most acid phosphatases, the purified enzyme showed a narrow range of substrate preferences, with the highest affinity towards thiamine phosphate (Km = 150 μM). Francisella species do not possess a thiamine biosynthetic pathway even though vitamin B1 is indispensable in numerous cellular functions. Consequently, thiamine should be incorporated from the environment, in this case, from the host cell. Our results suggested that AcpB could provide the hydrolytic activity necessary to transform the nontransportable phosphorylated vitamin B1 present in tissues to a form that can be absorbed by the intracellular pathogen. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Haloacid dehalogenase (HAD)-like hydrolases are a vast super- family of largely uncharacterized enzymes, with a few members shown to possess phosphatase, β-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 β-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. [ABSTRACT FROM AUTHOR]

To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. These screens identified the presence of nucleotidase activity in three uncharacterized E. coli proteins, SurE, YfbR, and YjjG, that belong to different enzyme super-families: SurE-like family, HD domain family (YfbR), and haloacid dehalogenase (HAD)-like superfamily (YjjG). The phosphatase activity of these proteins had a neutral pH optimum (pH 7.0–8.0) and was strictly dependent on the presence of divalent metal cations (SurE: Mn2+ > Co2+ > Ni2+ > Mg2+; YfbR: Co2+ > Mn2+ > Cu2+; YjjG: Mg2+ > Mn2+ > Co2+). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo- and deoxyribonucleoside 5′-monophosphates and ribonucleoside 3′-monophosphates with highest affinity to 3′-AMP. SurE also hydrolyzed polyphosphate (ex- opolyphosphatase activity) with the preference for short-chain-length substrates (P20–25). YfbR was strictly specific to deoxyribonucleoside 5′-monophosphates, whereas YjjG showed narrow specificity to 5′-dTMP, 5′- dUMP, and 5′-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di- and triphosphates: YfbR was equally sensitive to both di- and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli. [ABSTRACT FROM AUTHOR]

In all mature tRNAs, the 3′-terminal CCA sequence is synthesized or repaired by a template-independent nucleotidyltransferase (ATP(CTP):tRNA nucleotidyltransferase; EC 2.7.7.25). The Escherichia coli enzyme comprises two domains: an N-terminal domain containing the nucleotidyltransferase activity and an uncharacterized C-terminal HD domain. The HD motif defines a superfamily of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins and domains associated with nucleotidyltransferases and helicases from bacteria, archaea, and eukaryotes. The C-terminal HD domain in E. coli tRNA nucleotidyltransferase demonstrated Ni2+-dependent phosphatase activity toward pyrophosphate, canonical 5′-nucleoside tri- and diphosphates, NADP, and 2′-AMP. Assays with phosphodiesterase substrates revealed surprising metal-independent phosphodiesterase activity toward 2′,3′-cAMP, -cGMP, and -cCMP. Without metal or in the presence of Mg2+, the tRNA nucleotidyltransferase hydrolyzed 2′,3´-cyclic substrates with the formation of 2′-nucleotides, whereas in the presence of Ni2+, the protein also produced some 3′-nucleotides. Mutations at the conserved His-255 and Asp-256 residues comprising the C-terminal HD domain of this protein inactivated both phosphodiesterase and phosphatase activities, indicating that these activities are associated with the HD domain. Low concentrations of the E. coli tRNA (10 nM) had a strong inhibiting effect on both phosphatase and phosphodiesterase activities. The competitive character of inhibition by tRNA suggests that it might be a natural substrate for these activities. This inhibition was completely abolished by the addition of Mg2+, Mn2+, or Ca2+, but not Ni2+. The data suggest that the phosphohydrolase activities of the HD domain of the E. coli tRNA nucleotidyltransferase are involved in the repair of the 3′-CCA end of tRNA. [ABSTRACT FROM AUTHOR]