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

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 K and V were found to be 32 µM of HgCl 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. Cu 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 Hg bioremediation.

Indigeneous Streptomyces spp. isolated from Cyperus rotundus rhizosphere indicate high mercuric reductase activity as a pontential bioremediation agent

“…This is in line with Copeland (2000) which states that most enzymes work optimally at neutral pH. This statement was confirmed by Bafana et al (2017), Zeroual et al (2003), Olson et al (1982), Nakahara et al (1985) which states that the optimum mercury reductase at pH approaches neutral pH.…”

“…The molecular size of mercury reductase of several gram-positive and gram-negative bacteria that has been identified was ranged from 56 kDa and 62 kDa in Pseudomonas aeruginosa PAO 9501 (Fox and Walsh 1981), 60 kDa in Lysinibacillus sphaericus strain G1 (Bafana et al 2017), 69 kDa in Bacillus sp (Moore et al 1989), 54 kDa and 69 kDa in Azotobacter chroococcum (Gosh et al 1998), 64 kDa and 55.5 kDa in Escherichia coli J531 (R831) (Schottel 1977), and 62 kDa in Klebsiella pneumoniae (Zeroual et al 2003). Thus, it can be estimated the molecular weight of mercury reductase Streptomyces spp.…”

Purification, characterization, and crystal structure of YhdA‐type azoreductase from Bacillus velezensis

“…The purified enzyme was bright yellow colored, indicating the presence of a tightly bound flavin cofactor. The nature of flavin cofactor and enzyme: cofactor ratio were determined as reported earlier 16 …”

Thermal Stability of a Mercuric Reductase from the Red Sea Atlantis II Hot Brine Environment as Analyzed by Site-Directed Mutagenesis

“…Surprisingly, in comparison with both ATII-LCL-NH and ATI-LCL, the 2D (aspartic acid 415 and 416) mutation alone increased the thermostability of the molecule, as it retained 81% of its activity after 10 min of incubation at 70°C. Interestingly, the catalytic efficiency (k cat /K m ) of the heat-stable 2D mutant was much higher than that of the recently reported Metallosphaera sedula MerA (31) and Lysinibacillus sphaericus MerA (32), which makes it a better candidate for mercuric bioremediation.…”