A novel mercuric reductase from the unique deep brine environment of Atlantis II in the Red Sea

Abstract

A unique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg(2+), and efficiently detoxifies Hg(2+) in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL.

Keywords: Atlantis II Brine Pool; Enzyme Kinetics; Enzyme Mechanisms; Enzyme Structure; Extreme Halophilic; Mercuric Reductase; Metagenomics; Mutagenesis Site-specific; Red Sea Atlantis II; Thermophilic.

FIGURE 1.

Alignment of the amino acid sequences of MerA ATII-LCL and the soil ortholog. The sequence of the soil enzyme is shown in bold and that of the ATII-LCL in light gray; the NmerA domain is underlined in black; the dimerization domain that is conserved among the homodimeric pyridine nucleotide-disulfide oxidoreductases is underlined in red; and the β-strand structures present in the dimerization domain are overlined in blue. The cysteine pairs 11/14 and 558/559 involved in binding of Hg²⁺, and cysteines 136/141, which form the disulfide bridge involved in Hg²⁺ reduction, are highlighted in yellow; negatively charged substitutions in MerA ATII-LCL are shown in red. The two sequences that contribute to thermostability are boxed in green (box1 and box2). The acidic residues marked M in the ATII-LCL sequence were replaced by the corresponding amino acids in the soil enzyme in the indicated ATII-LCL mutants.

FIGURE 2.

Patterns of amino acid substitutions present in the ATII-LCL MerA relative to the soil ortholog. A, amino acid compositions of the ATII-LCL and soil enzymes were compared, and the plot shows the net change in the number of each of the listed amino acids in the ATII-LCL enzyme. B, frequency of substitutions in ATII-LCL plotted against corresponding residue in the soil enzyme. C, patterns of gain, loss, and net change of amino acids in ATII-LCL relative to the soil enzyme based on their functional classifications.

FIGURE 3.

Effects of temperature, salt, and mercury concentration on the catalytic activities of ATII-LCL and soil MerAs. A, thermostability. The ATII-LCL and soil MerAs were incubated at the indicated temperature for 10 min, and the residual enzymatic activities were assayed under standard conditions. B, effect of NaCl concentration on MerA activity. The enzymatic activities were measured in reaction mixtures containing the indicated concentrations of NaCl. C, sensitivity to HgCl₂. The enzymes were incubated in the assay mixture for 10 min, in the absence of NADPH and presence of the indicated concentrations of HgCl₂. The enzyme activities were then measured by addition of NADPH to the assay mixture. D, determination of the MIC for HgCl₂. Solutions containing increasing concentrations of HgCl₂ were placed in wells punched in LB-agar plates (supplemented with 50 μg/ml kanamycin and 1 mm isopropyl β-d-thiogalactoside) inoculated with E. coli transformants. The plates were incubated for 24 h at 37 °C. The radii of the clear zones are a measure of the toxic effect of HgCl₂ on bacterial growth.

FIGURE 4.

Properties of the ATII-LCL MerA mutants. Mutants generated by site-directed mutagenesis (M1, M4, M15, M16, the double mutant M15/M16, M17, and M18) were examined as described in Fig. 4. Refer to Table 1 for details regarding each mutant.

FIGURE 5.

Three-dimensional structure models of the homodimer ATII-LCL MerA. Homology modeling based on the Tn501 Mer reductase (12) was carried out as described under “Experimental Procedures.” Front and top views are presented. Yellow spheres denote the sulfur atoms of the cysteines involved in Hg²⁺ binding and reduction; red spheres represent the side-chain carboxylic oxygens of glutamic acid residues that were mutated in this work. A shows the C- and N-terminal domains portrayed in schematic form; one subunit is shown in dark blue and the other in cyan. Box1 (random coiled loop) and box2 (β-strand) are highlighted in green. B highlights the disposition of box1 and box2 and the functionally important cysteine pairs and nearby glutamic acid residues, which are labeled in C according to the subunit to which they belong (see supplemental Fig. 3 for the structure of the monomer).

FIGURE 6.

Summary of the proposed functions of glutamic acid residues that affected the catalytic properties and the halophilicity of ATII-LCL MerA.