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 being employed to predict function, including sequence similarity searches, analysis of gene expression, protein interaction, and protein structure. Enzymes comprise a group of target proteins that require experimental characterization for accurate functional annotations. Here I applied enzyme genomics to identify new enzymes by screening individually purified proteins for enzymatic activity under relaxed reaction conditions, which allowed me to identify the subclass or sub-subclasses of enzymes to which the unknown protein belongs. Further biochemical characterization of proteins was facilitated by the application of secondary screens with natural substrates (substrate profiling). Application of general enzymatic screens and substrate profiling greatly sped up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches. As a test case, I used this approach to characterize the members of the haloacid dehalogenase (HAD)-like hydrolase superfamily, which consists mainly of uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Low sequence similarity between the members of the HAD superfamily precludes the computational prediction of their substrates and functions. Using a representative set of 80 phosphorylated substrates I characterized the phosphatase activities of 21 soluble HADs from Escherichia coli and seven soluble HADs from Saccharomyces cerevisiae. E. coli HADs show broad and overlapping substrate specificity against a wide range of phosphorylated metabolites. The yeast enzymes were more specific, and one protein also showed protein phosphatase activity. Comparison of HAD substrate profiles from two model organisms showed several “functional niches” that are occupied by HADs, which include hydrolysis of nucleotides, phosphoglycolate, phosphoserine, and pyridoxal phosphate. I proposed the cellular function for a number of HADs from both organisms based on substrate specificities. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, E. coli YniC.

ANGLICKÝ ABSTRAKT Functional study of the putative nucleotidase encoded by spr1057 gene in Streptococcus pneumoniae, a likely homolog of Escherichia coli protein YjjG. Bacterial cells are constantly exposed to innumerable toxic substances, either in their external environment or by by-products of their own metabolism. For these reasons, the bacterial cells evolved several mechanisms to cope with this challenge. These mechanisms are represented by: blocking the uptake, export by specific transporters as well as specific inactivation of these substance by enzymes. A particular group of these toxic substances are noncanonica nucleotides, which can directly inhibit bacterial cell DNA replication or can result in increased mutation rate. Enzymes recognizing these modified derivatives are known as "house-cleaning" nucleotide phsphateses, which can inactivate the potentially mutagenic nucleotides and prevent their incorporation into DNA and RNA. Some of the "house- cleaning" enzymes belong to a group of haloacid dehalogenase enzymes (haloacid dehalogenase-like hydrolase superfamily), which are found in many bacterial species. This thesis is focused on the function of hypothetical protein Spr1057 of Streptococcus pneumoniae with an unknown function. Sequence comparison revealed that Spr1057 has a significant...