Biochemical and Structural Properties of a Thermostable Mercuric Ion Reductase from Metallosphaera sedula

Abstract

Mercuric ion reductase (MerA), a mercury detoxification enzyme, has been tuned by evolution to have high specificity for mercuric ions (Hg(2+)) and to catalyze their reduction to a more volatile, less toxic elemental form. Here, we present a biochemical and structural characterization of MerA from the thermophilic crenarchaeon Metallosphaera sedula. MerA from M. sedula is a thermostable enzyme, and remains active after extended incubation at 97°C. At 37°C, the NADPH oxidation-linked Hg(2+) reduction specific activity was found to be 1.9 μmol/min⋅mg, increasing to 3.1 μmol/min⋅mg at 70°C. M. sedula MerA crystals were obtained and the structure was solved to 1.6 Å, representing the first solved crystal structure of a thermophilic MerA. Comparison of both the crystal structure and amino acid sequence of MerA from M. sedula to mesophillic counterparts provides new insights into the structural determinants that underpin the thermal stability of the enzyme.

Keywords: MerA; biosensor; mercuric reductase; mercury detoxification; structure; thermophile; thermostability.

Figure 1

Maximum-likelihood phylogenetic reconstruction of MerAs, with homologs from thermophilic taxa highlighted in red. MseMerA is boldfaced. Bootstrap support is indicated by black (>90), gray (80–89), and open (70–79) circles. Nodes with no symbol exhibited bootstrap values of <70.

Figure 2

MseMerA aligned with other MerAs reveals two loop regions, L1 and L2, which may be involved in conferring thermostability, and two positions at 326 and 452 (highlighted with stars), where the active site region is different between thermophiles and mesophiles.

Figure 3

(A) Structural superimposition of MseMerA monomer (cyan) with Tn501MerA (green) reveals a decrease in loop regions (labeled L1 and L2) in MseMerA. (B) Cartoon representation of a dimer of MseMerA with bound FAD.

Figure 4

NADPH oxidation activity of MseMerA incubated at temperatures ranging from 37 to 70°C.

Figure 5

(A) Structural superimposition of MseMerA with Tn501MerA 1ZK7 shows the Y441′/V317 amino acids conserved in mesophiles and the F441/E317 amino acids conserved in thermophiles, suggesting an alternative Hg²⁺ coordination strategy in MseMerA. (B) An alternative angle of the active site environment of MseMerA superimposed onto Tn501MerA 4K7Z, which depicts the Hg²⁺ ion bound to the c-terminal cysteines. The monomer with the C-terminal cysteines is noted by a “prime” designation.