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Background: Acetylation and efflux of carbohydrates during cellular metabolism is a well-described phenomenon associated with a detoxification process to prevent metabolic congestion. It is still unclear why cells discard important metabolizable energy sources in the form of acetylated compounds., Methods: We describe the purification and characterization of an approximately 28-kDa intracellular carboxylesterase (YjfP) and the analysis of gene and protein expression by qRT-PCR and Western blot., Results: qRT-PCR and Western blot, respectively, showed that yjfP is upregulated during the diauxic lag in cells growing with a mixture of glucose and lactose. The β-galactosidase activity in the ΔyjfP strain was both delayed and half the magnitude of that of the wild-type strain. YjfP-hyperproducing strains displayed a long lag phase when cultured with glucose and then challenged to grow with lactose or galactose as the sole carbon source., Conclusion: Our results suggest that YjfP controls the intracellular concentration of acetyl sugars by redirecting them to the main metabolic circuits. Instead of detoxification, we propose that sugar acetylation is utilized by the cell for protection and to prevent the metabolism of a necessary minimal intracellular sugar pool. Those sugars can eventually be exported as a side effect of these mechanisms., (© 2016 S. Karger AG, Basel.)

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.

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.

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.

Background: Acetylation and efflux of carbohydrates during cellular metabolism is a well-described phenomenon associated with a detoxification process to prevent metabolic congestion. It is still unclear why cells discard important metabolizable energy sources in the form of acetylated compounds. Methods: We describe the purification and characterization of an approximately 28-kDa intracellular carboxylesterase (YjfP) and the analysis of gene and protein expression by qRT-PCR and Western blot. Results: qRT-PCR and Western blot, respectively, showed that yjfP is upregulated during the diauxic lag in cells growing with a mixture of glucose and lactose. The β-galactosidase activity in the ΔyjfP strain was both delayed and half the magnitude of that of the wild-type strain. YjfP-hyperproducing strains displayed a long lag phase when cultured with glucose and then challenged to grow with lactose or galactose as the sole carbon source. Conclusion: Our results suggest that YjfP controls the intracellular concentration of acetyl sugars by redirecting them to the main metabolic circuits. Instead of detoxification, we propose that sugar acetylation is utilized by the cell for protection and to prevent the metabolism of a necessary minimal intracellular sugar pool. Those sugars can eventually be exported as a side effect of these mechanisms. © 2016 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]