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Structural and functional characterization of mercuric reductase from Lysinibacillus sphaericus strain G1

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Abstract

In response to the widespread presence of inorganic Hg in the environment, bacteria have evolved resistance systems with mercuric reductase (MerA) as the key enzyme. MerA enzymes have still not been well characterized from gram positive bacteria. Current study reports physico-chemical, kinetic and structural characterization of MerA from a multiple heavy metal resistant strain of Lysinibacillus sphaericus, and discusses its implications in bioremediation application. The enzyme was homodimeric with subunit molecular weight of about 60 kDa. The Km and Vmax were found to be 32 µM of HgCl2 and 18 units/mg respectively. The enzyme activity was enhanced by β-mercaptoethanol and NaCl up to concentrations of 500 µM and 100 mM respectively, followed by inhibition at higher concentrations. The enzyme showed maximum activity in the pH range of 7–7.5 and temperature range of 25–50 °C, with melting temperature of 67 °C. Cu2+ exhibited pronounced inhibition of the enzyme with mixed inhibition pattern. The enzyme contained FAD as the prosthetic group and used NADPH as the preferred electron donor, but it showed slight activity with NADH as well. Structural characterization was carried out by circular dichroism spectrophotometry and X-ray crystallography. X-ray confirmed the homodimeric structure of enzyme and gave an insight on the residues involved in catalytic binding. In conclusion, the investigated enzyme showed higher catalytic efficiency, temperature stability and salt tolerance as compared to MerA enzymes from other mesophiles. Therefore, it is proposed to be a promising candidate for Hg2+ bioremediation.

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Acknowledgements

Amit Bafana is thankful to Indian National Science Academy (INSA), India, for the award of INSA Visiting Fellowship (No. SP/VF-30/2014-15) to carry out part of this study at Indian Institute of Science, India.

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Authors and Affiliations

  1. Environmental Impact and Sustainability Division, CSIR-NEERI (National Environmental Engineering Research Institute), Nagpur, 440020, India

    Amit Bafana

  2. Molecular Biophysics Unit, Indian Institute of Science, Bangalore, 560012, India

    Farha Khan & Kaza Suguna

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  1. Amit Bafana
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  2. Farha Khan
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  3. Kaza Suguna
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Correspondence to Amit Bafana.

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Supplementary Fig. S1

Gel filtration chromatography for estimation of MerA native molecular weight. The calibration proteins were thyroglobulin—670 kDa, γ-globulin—158 kDa, ovalbumin—44 kDa, myoglobin—17 kDa, and vitamin B12—1.3 kDa. Arrow represents elution of MerA (JPEG 25 kb)

Supplementary Fig. S2

Fluorescence-based thermal shift assay for MerA using SYPRO Orange. Black line represents the denaturation curve, while red line shows modeled curve (JPEG 30 kb)

Supplementary Fig. S3

Determination of MerA secondary structure by circular dichroism spectrometry. Based on the predicted spectrum, MerA contained 33.75% α helix and 15.49% β strand (JPEG 30 kb)

Supplementary Fig. S4

Sequence alignment of L. sphaericus MerA enzyme with homologs. Homologous proteins are indicated by their PDB id, while the L. sphaericus enzyme is shown as merA. Identical residues are shaded with red background, while similar residues are shown in blue box (PDF 21 kb)

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Bafana, A., Khan, F. & Suguna, K. Structural and functional characterization of mercuric reductase from Lysinibacillus sphaericus strain G1. Biometals 30, 809–819 (2017). https://doi.org/10.1007/s10534-017-0050-x

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  • DOI: https://doi.org/10.1007/s10534-017-0050-x

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