Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine. In each organism, one glutaminase showed higher affinity to glutamine ( E. coli YbaS and B. subtilis YlaM; K m 7.3 and 7.6 mM, respectively) than the second glutaminase ( E. coli YneH and B. subtilis YbgJ; K m 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical beta-lactamase-like fold and conservation of several key catalytic residues of beta-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme-glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.
The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.
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.
