Part:BBa_K2194000

chrR6 Chromate Reductase Enzyme

The chrR6 protein is a mutated/enhanced version (^Tyr128^Asn) of the chrR enzyme originally found in Escherichia coli. It has chromate and uranyl reductase activity. BioBrick BBa_K2194000 contains only the cds for this protein.

Biology and Usage

The chrR6 enzyme is a mutated and enhanced version (^Tyr128^Asn) of the soluble chrR enzyme originally found in Escherichia coli and Pseudomonas putida. It has chromate and uranyl reductase activity. The enzyme chrR6 reduces hexavalent chromium (Cr(VI)) directly to trivalent chromium (Cr(III)) without producing an unstable pentavalent chromium (Cr(V)) intermediate and minimizing generation of toxic reactive oxygen species. [1]

As determined from studies on purified enzymes [1], ^Tyr128^Asn substitution increases the rate of Cr(VI) reduction from 295 ± 27 nmol mg protein^-1 min^-1, as observed for chrR, to 8,812 ± 611 nmol mg protein^-1 min^-1. Observed K_cat/K_m values are 4.5 × 10⁴ ± 3 × 10³ for chrR and 1.3 × 10⁷ ± 3 × 10⁵ for chrR6 [1]. When the activity of chrR6 and chrR was studied in vivo in soil bacteria P. putida, no enhanced chromium or uranium reduction activity was observed for chrR6 transformed bacteria, which led to the conclusion that limited cell permeability to Cr(VI) prevents the reduction activity.

Figure 1: Rate of Cr(VI) reduction by E. coli MG1655 transformed with chrR6 and sulfate transport system cysPUWA.

Figure 1 compares the Cr(VI) reduction rate standardized for OD₆₀₀ of E. coli MG1655 versus E. coli MG1655 cotransformed with the sulfate transporter system (BBa_K2194004) and constitutively expressed chrR6 reductase enzyme (BBa_K2194000). The data indicates that below an initial [Cr(VI)] of 80 uM the cotransformed bacteria are more efficient at reducing Cr(VI) than wild type bacteria. Above 80 uM initial [Cr(VI)], the trend reverses and wild type bacteria are more efficient. This supports the hypothesis that increasing cell permeability to chromate (in this case via sulfate transporters) increases the reduction efficiency of chrR6, but that after a threshold concentration the increased permeability becomes toxic for the cell.

Figure 2: the amount of Cr(VI) reduced by E. coli MG1655 transformed with chrR6 and cysPUWA wild-type E. coli

Figure 2 presents the same data as Figure 1 but as an amount of total Cr(VI) reduced rather than the OD₆₀₀ standardized rate. (The x-axis and the light blue bars display the initial concentration of Cr(VI) and the y-axis displays the final concentration of Cr(VI). Comparison of the dark blue bars to the light blue bar at each different initial concentration of Cr(VI) reveals the total change in [Cr(VI)].

[1] Barak, Y. et al. “Analysis of Novel Soluble Chromate and Uranyl Reductases and Generation of an Improved Enzyme by Directed Evolution.” Applied and Environmental Microbiology 72.11 (2006): 7074–7082. Web.
[2] Robins, Katherine J. et al. “Escherichia Coli Nema Is an Efficient Chromate Reductase That Can Be Biologically Immobilized to Provide a Cell Free System for Remediation of Hexavalent Chromium.” PLoS ONE (2013): n. pag. Web.
[3] Cheung, K. H., and Ji Dong Gu. “Mechanism of Hexavalent Chromium Detoxification by Microorganisms and Bioremediation Application Potential: A Review.” International Biodeterioration and Biodegradation 59.1 (2007): 8–15. Web.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 570
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 337
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3
  Illegal BsaI.rc site found at 583