Primer período: del Formativo a finales del Desarrollo Regional (hasta 700 d.C.)

5.3.1 Proposed formaldehyde binding mechanism

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Figure 5. 2 frmRAPro (10 nM) was anaerobically titrated with FrmR in the presence of 5 mM

EDTA and in the presence of 20 µM of (A) acetaldehyde and (B) ethanol. DNA-binding was monitored by fluorescence anisotropy. Data were fit to a model describing a 2:1 FrmR tetramer (non-dissociable):DNA stoichiometry.

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Figure 5. 3 A In vivo expression from PfrmRA-frmR::lacZ. Representative (n=3) β-galactosidase

activity following growth of SL1344ΔfrmR, harbouring either PfrmRA::lacZ or PfrmRA-frmR::lacZ

construct, in M9 minimal media in the absence or presence of minimum non-inhibitory concentrations of formaldehyde. B In vivo expression from PfrmRA-frmR::lacZ Representative

(n>3) β-galactosidase activity following growth of SL1344ΔfrmR, harbouring a PfrmRA-

frmR::lacZ construct, in M9 minimal media in the absence or presence of minimum non-

inhibitory concentrations of ethanol, methanol, 1-butanol, 1-propanol, 2-propanol, formaldehyde and acetaldehyde. These data were obtained by Dr. Deenah Osman.

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the conformational change that causes the release of its promoter region involves a cysteine (Higgins & Giedroc 2014; Law 2012). Cysteine residues are involved in sensing in other RcnR/CsoR family members (Iwig & Chivers 2009; Foster et al. 2012; Ma et al. 2009a). Moreover, reactions of thiols with carbonyl compounds are well known (Jencks 1969; Kallen 1971; Lienhard & Jencks 1966). In particular, the reaction of cysteine residues with formaldehyde can be divided in two steps. First, the sulphur atom on the thiol group attacks the carbonyl group of formaldehyde producing a hemithioacetal intermediate, followed by removal of one molecule of water and ring closure due to the attack of the amino group from cysteine backbone to yield a thiazolidinecarboxylic acid (Schubert 1935; Ratner & Clarke 1937).

Aqueous formaldehyde is present in its hydrated form, methanediol (CH2(OH)2); however,

carboxyl compounds are reactive toward nucleophilic attack in their unhydrated forms (Lienhard & Jencks 1966; Sander & Jencks 1968; Bell & Evans 1966; Bell 1966; Kallen & Jencks 1966). Formaldehyde dehydration is catalyzed by hydroxide ions above pH = 7.0 (Le Henaff 1960) and was shown to not be a rate determining step in the subsequent reactions with cysteine residues (Kallen 1971).

A conserved residue, Cys35, was identified as the cysteine that reacts with formaldehyde in E.

coli FrmR (Law 2012), and coincides with the only cysteine residue (located on helix 2,

monomer 1) found in Salmonella FrmR. Figure 5. 4 outlines the proposed reaction mechanism between Salmonella FrmR and formaldehyde when this is present in low concentrations. In order to generate the reactive thiolate group (deprotonated form of thiol) on Cys35 a nearby residue is needed to act as a Lewis base and remove the proton (H+) from the sulfhydryl group. This residue, herein called amino acid X, has not been identified but good candidates could be His60 or Asp63 since both have a carboxylate group deprotonated at pH values close to 7.0 (pKr

His

= 6.04 and pKr Asp

= 3.90 in aqueous solution at 20 ᵒC). The now negatively charged sulphur atom of the Cys35 thiol group can give a nucleophilic addition on the formaldehyde yielding a tetrahedral intermediate. Subsequent abstraction of a proton from amino acid X (restoring the initial deprotonated form) yields an alcohol group. It has been postulated that a second residue from the N-terminal region of a different monomer is involved in this mechanism using its amino group (Higgins & Giedroc 2014). The amino terminus region is also required for metal-sensing by E. coli RcnR (Iwig et al. 2008; Higgins et al. 2012b). We here suggest that this residue could be Pro2 (on helix 1, monomer 2) which retains a basic pyrrolidine ring.

When a sample of purified FrmR was examined by quantitative amino acid analysis, the result was consistent with the presence of only three methionine residues instead of four, as the theoretical sequence would predict. Results from the Amino Acid Analysis performed on the protein are shown in the Appendix. If the number of moles per ml of Met, 513, is divided by the number of moles per ml of Tyr (since FrmR possesses only one tyrosine), 181, the resulting

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A.

A.

Figure 5. 4 Proposed mechanism of action of formaldehyde crosslinking FrmR. The reaction takes place in several steps. An amino acid (possibly His60 or Asp63, herein called amino acid X) close to Cys35 (helix 2, monomer 1) deprotonates the sulphur atom of the Cys35 thiol group, placing a negative charge on sulphur and making the thiol group more nucleophilic. Subsequent nucleophilic addition on the formaldehyde by the lone-pair electrons of the thiol on the Cys35 yields a tetrahedral intermediate. The basic intermediate abstracts a proton (H+) from amino acid X to yield an alcohol group and regenerate the deprotonated form of amino acid X. Pro2 on helix 1, monomer 2 is the first residue of the N-terminal region and possesses a pirrolydine ring. Pyrrolidine is a strong base and it is among the most basic simple amines in nature (its conjugate acid has pKa = 11.27) (Hall 1957).The nitrogen on this residue attacks the positively polarized carbon. The alcohol group abstracts the proton from the now positively charged nitrogen and acts as a good living group (H2O) to yield the final product.

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value, 2.83, can be rounded up to 3. The analysis has been repeated twice on different preparations of FrmR, confirming the detection of only three methionine residues. This outcome was interpreted as evidence of the cleavage of Met1, a co-translational process predicted to occur in the ~ 80 % of the totality of proteins (Waller 1963; Matheson et al. 1975; Brown 1970; Frottin et al. 2006). Moreover, the cleavage of Met1 was confirmed by LC-MS analysis, performed by our collaborators Dr. Huggins and Dr. Chen (Procter and Gamble Mason Business Centre, Cincinnati, Ohio), on FrmR and E64HFrmR samples following digestion with trypsin (0.5 mg ml-1) to obtain specific transitions (Figure 8. 3, Appendix). The presence of both PHSPEDK (where Met1 has been cleaved) and MPHSPEDK peptides was detected, but the qualitative nature of the analysis did not inform of their proportions. However, the presence of MPHSPEDK can be explained by considering that purified proteins are routinely overexpressed up to ≥ 5 mg/ml and the activity of the methionine aminopeptidase, the enzyme designated to cleave the methionine, would be hence limited by the large amount of available substrate. Since the Amino Acid Analysis previously discussed did not detect a fourth methionine, it is plausible to assume that a substantial proportion of the purified protein has undergone the cleavage. This result is in accord with what was observed for E. coli FrmR by Law (Law 2012). The main FrmR peak detected by time-of-flight mass-spectrometry (TOF-MS) (ionization source: electrospray) has a molecular mass of 10186 Da instead of 10318 Da (the predicted FrmR molecular mass) (Law 2012). The difference between the predicted and the experimental mass is 132 Da which coincides with the cleavage of the methionine (131 Da).

In this scenario Pro2 is the first residue of the N-terminal region and it possesses a secondary amine group not involved in peptide bonding. According to the mechanism proposed here, the nitrogen on this residue attacks the electrophilic carbon on the tetrahedral intermediate and the alcohol group abstracts the proton from the now positively charged nitrogen creating a good leaving group (H2O) to yield the final product. The specific crosslink would connect monomer 1 to monomer 2 of tetrameric FrmR causing a conformational rearrangement that produces a new protein assembly with a lower DNA-binding affinity.

In order to further explore these hypotheses, DNA-binding affinities of mutant FrmR proteins in the absence and in presence of stoichiometric concentration of formaldehyde were analysed.