Molecular Basis of Formaldehyde Detoxification

Abstract

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 ␣-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the ). 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.Formaldehyde is an extremely reactive chemical producing covalently cross-linked complexes with proteins and nucleic acids (1-3). It is a common environmental contaminant found in many industrial and medical products, as well as being endogenously produced in all living organisms as a result of metabolism (methionine, histamine, methanol, and methylamine), spontaneous dissociation of 5,10-mehylene tetrahydrofolate, or oxidative demethylation of DNA and RNA (4 -9). To prevent the lethal and mutagenic effects of formaldehyde, several repair mechanisms have evolved. Detoxification of formaldehyde can be carried out by enzymes like formaldehyde dismutase, methylformate synthase, or glutathioneindependent formaldehyde dehydrogenase (10 -12). However, these enzymes have been found in only a limited number of organisms, whereas a glutathione-dependent repair system appears to be widespread in nature and has been found in most prokaryotes (except for archaea) and all eukaryotes (7,(13)(14)(15).In this process (shown in Scheme 1), formaldehyde spontaneously reacts with GSH to produce S-hydroxymethylglutathione, which is then oxidized by formaldehyde dehydrogenase to S-formylglutathione. Finally, this compound is hydrolyzed to formate and GSH by S-formylglutathione hydrolase (FGH).2 These functionally related enzymes, formaldehyde dehydrogenase and FGH, also show genetic linkage, since their genes are adjacent in many bacterial genomes or even fused in some eukaryotes (16).Formaldehyde dehydrogenases (class III alcohol dehydrogenases) were extensively characterized both structurally and biochemically (17-20), whereas FGHs have received far less attention. Only three eukaryotic FGHs (from the human liver, Saccharomyces cerevisiae, and Arabidopsis thaliana) (21-23) have been purified and partially characterized, but the homologous enzyme from bacteria has not been yet purified. The FGH enzymes from yeasts and A. thaliana are not strictly specific to S-formylglutathione and also show significant carboxylesterase activity against model substrates ␣-n...

Structure-Based Mutational Studies of Substrate Inhibition of Betaine Aldehyde Dehydrogenase BetB from Staphylococcus aureus

“…The genes encoding S. aureus BetB (GenBank 3237065, Uniprot ID Q5HCU0), and E. coli YdcW (GenBank 945876, Uniprot ID P77674) were PCR amplified using S. aureus and E. coli genomic DNA and cloned into the IPTG (isopropyl-␤-Dthiogalactopyranoside)-inducible protein expression vector p15TV-L as described previously (36). The resulting plasmids were transformed into the E. coli BL21(DE3) Gold strain (Stratagene, USA), and the recombinant proteins were purified using affinity chromatography on Ni-nitrilotriacetic acid (Ni-NTA) resin (Qiagen, Germany).…”

Inhibition of AcpA Phosphatase Activity with Ascorbate Attenuates Francisella tularensis Intramacrophage Survival

“…Under these conditions, enzyme activity was linear with time for at least 60 min. The amount of p-nitrophenol released was quantified at A 412 using the extinction coefficient ⑀ ϭ 16,300 M Ϫ1 cm Ϫ1 as described previously (16). Enzymatic activity was also determined using 2-phosphoascorbate as a substrate using malachite green ammonium molybdate detection reagent (17).…”

An Internal Reaction Chamber in Dimethylglycine Oxidase Provides Efficient Protection from Exposure to Toxic Formaldehyde

“…An in vitro reporter system for formaldehyde was created using fusions of the gfp gene and the E. coli frm operon (23,24). A UV light-sensitive version of the gfp gene (25) optimized for prokaryotic expression was used (Clontech).…”

“…Bacteria and Growth Conditions-E. coli DH5␣ was routinely used as the containment strain in all clonings. Experiments detecting the in vivo formation of formaldehyde were done using a formaldehyde-sensitive strain, E. coli W3GM (24). Bacteria were either grown on Luria-Bertani medium (26) or on minimal medium (27) with 10 mM glucose as the sole source of carbon and energy.…”

“…The E. coli formaldehyde detoxification system contains a putative formaldehyde sensor, FrmR, which regulates expression of the glutathione-dependent formaldehyde dehydrogenase gene, frmA, and the putative S-formylglutathione hydrolase gene, frmB (23,24). We fused a UV light-sensitive version of gfp optimized for prokaryotic expression (25) to the E. coli promoter region of frmR and cloned this into pET-or pColabased plasmid vectors.…”