Molecular dissection of a dedicated formaldehyde dehydrogenase from Mycobacterium smegmatis

Abstract

Accumulation of formaldehyde, a highly reactive molecule, in the cell is toxic, and requires detoxification for the organism's survival. Mycothiol-dependent formaldehyde dehydrogenase or S-nitrosomycothiol reductase (MscR) from Mycobacterium smegmatis and Mycobacterium tuberculosis was previously known for detoxifying formaldehyde and protecting the cell against nitrosative stress. We here show that M. smegmatis MscR exhibits a mycothiol-independent formaldehyde dehydrogenase (FDH) activity in vitro. Presence of zinc in the reaction enhances MscR activity, thus making it a zinc-dependent FDH. Interestingly, MscR utilizes only formaldehyde and no other primary aldehydes as its substrate in vitro, and M. smegmatis lacking mscR (ΔmscR) shows sensitivity exclusively toward formaldehyde. Bioinformatics analysis of MscRs from various bacteria reveals 10 positionally conserved cysteines, whose importance in structural stability and biological activity is not yet investigated. To explore the significance of these cysteines, we generated MscR single Cys variants by systematically replacing each cysteine with serine. All of the Cys variants except C39S and C309S are unable to show a complete rescue of ΔmscR on formaldehyde, show a significant loss of enzymatic activity in vitro, pronounced structural alterations as probed by circular dichroism, and loss of homotetramerization on size exclusion chromatography. Our data thus reveal the importance of intact cysteines in the structural stability and biological activity of MscR, which is a dedicated FDH in M. smegmatis, and shows ~84% identity with M. tuberculosis MscR. We believe that this knowledge will further help in the development of FDH as a potential drug target against M. tuberculosis infections.

Keywords: M. smegmatis; conserved cysteine; cysteine mutants; formaldehyde stress; mycothiol-dependent formaldehyde dehydrogenase; site directed mutagenesis.

FIGURE 1

MscR is a Zn‐dependent homotetrameric formaldehyde dehydrogenase. (a) The plot shows the specific activity (SA) of purified MscR with formaldehyde (FA), acetaldehyde (AA), and propionaldehyde (PA) as substrates. (b) The SA of purified MscR with formaldehyde as substrate in the presence (+Zn) and absence (−Zn) of Zn²⁺. In both panels (a) and (b), SA is plotted as μmol per min per mg of MscR protein. (c) The graph shows the size exclusion chromatography data obtained by plotting the elution volume (V _e in ml) and the absorbance of protein at 280 nm (A₂₈₀). The purified native MscR (wildtype [WT]) with a monomeric molecular weight of 39.3 kDa elutes with an apparent molecular weight of ~158 kDa. For column calibration, known molecular weight proteins (thyroglobulin, 669 kDa; ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa) are used with their molecular weights marked in the graph. In both panels (a) and (b), the data represent an average of at least three independent experiments with error bars denoting SD. “****,” p‐value <.0001

FIGURE 2

MscR is a dedicated formaldehyde dehydrogenase and its overexpression leads to enhanced tolerance in Mycobacterium smegmatis. (a) Spot assay of wildtype M. smegmatis (WT), mscR knockout (ΔmscR), and the complemented strain (ΔmscR ^C ) in the absence (−ALD) and presence of various aldehydes such as formaldehyde (+FA), acetaldehyde (+AA), and propionaldehyde (+PA) is shown. The triangle above each image represents the serial dilution. (b) Residual formaldehyde levels present in the culture medium at different time intervals are plotted for the M. smegmatis wildtype (WT), mscR knockout (ΔmscR), and the complemented strain (ΔmscR ^C ) during their growth upon addition of formaldehyde. Control represents media with formaldehyde but without any cells. The data are a representation of an average of three independent experiments with error bars indicating SD. “***,” p‐value <.0002; “****,” p‐value <.0001; ns—not significant. (c) The graph shows the growth of mycobacterial strains such as M. smegmatis (WT), mscR knockout (ΔmscR), and the complemented strain (ΔmscR ^C ) measured in the absence (0 mM) and presence of 2 mM formaldehyde by monitoring the culture optical density at 600 nm (OD₆₀₀) at different time intervals

FIGURE 3

Comparison of protein sequences of mycothiol‐dependent formaldehyde dehydrogenases from various bacteria. The figure represents a weblogo, generated for all the cysteine‐containing regions present in the bacterial mycothiol dependent formaldehyde dehydrogenases. The y‐axis shows the relative frequency of the occurrence of amino acid at that position; a value of four bits represents 100% conservation. The x‐axis shows the position of the amino acid with respect to Mycobacterium smegmatis MscR. The position of cysteine in each case is marked with an asterisk (*)

FIGURE 4

Importance of conserved cysteine residues in the phenotypic rescue of mscR knock‐out and the catalytic activity of the enzyme. (a) Panel shows the spot assay performed for the wildtype Mycobacterium smegmatis (WT), mscR knockout (ΔmscR), and the knockout complemented with either WT MscR (ΔmscR ^C ) or cysteine‐substituted proteins such as C39S, C42S, C93S, C96S, C99S, C107S, C145S, C162S, C188S, and C309S, as indicated, on MB7H9‐agar plates in the absence (−FA) and presence (+FA) of exogenously‐added 1 mM formaldehyde. The triangle above each image represents the serial dilution. (b) The plot represents the specific activity (SA) of MscR and its Cys‐substituted versions in the presence of formaldehyde. The data represent an average of at least three independent experiments with error bars denoting SD. “****,” p‐value <.0001; ns—not significant

FIGURE 5

Effect of mutation of cysteines on the secondary structure of MscR. Both panels (a) and (b) show the far‐UV circular dichroism spectra of purified MscR protein and its Cys mutants. The plots show the molar ellipticity (ME) with respect to wavelength. The experiment was repeated multiple times; only one representative graph is shown here. The profiles are distributed between two graphs for clarity purpose, with WT present in both panels for comparison

FIGURE 6

Importance of cysteine residues in the oligomerization of MscR. Both panels (a) and (b) show the elution volume (Ve) of the wildtype (WT) MscR and its Cys variants; the profiles are distributed between two graphs for clarity purpose, with WT present in both panels for comparison. For calibration (calibrants), known molecular weight proteins (thyroglobulin, 669 kDa; ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; ovalbumin, 43 kDa) are used with their molecular weights marked in the graph. The experiment was repeated at least twice; only one representative image is shown here