Part:BBa_K5115063
hox and hyp, with EP targeted hoxF
Contents
Introduction
The Ni-Fe hydrogenase we use is an enzyme that functions in vivo bidirectionally for NAD+ reduction and NADH oxidation coupled to H2 uptake and H2 production, respectively. [1] In our design, the Ni-Fe hydrogenase works mainly to restore the nickel to a zero valence, which can help reduce nickel toxicity and collect nickel particles.
Usage and Biology
The Ni-Fe hydrogenase is made up of six major and three auxiliary subunits. The former includes hoxF, hoxU, hoxY, hoxH, hoxW and hoxI, while the latter includes hypA, hypB and hypF.
The hoxF and the hoxU form the module of NADH dehydrogenase. The hoxF is a hydrogenase subunit responsible for electron transport. The most important group in hoxF is FMN-b, which has the ability of switching electron. Under anaerobic conditions, NADH is oxidized to NAD+ on the surface of hoxF subunit. In the meanwhile, the electrons generated in this reaction travel through a series of processes to the hoxH, completing the reduction of the hydrogen ion. Under aerobic conditions, NAD+ is reduced to NADH on the surface of the hoxF subunit. The electron transferring is contrary to former. [2] The hoxU houses a 2Fe-2S cluster and is responsible for the role of conducting electrons between hoxH and hoxF. [3]
The hoxY and the hoxH form the module of catalytic center.The hoxY houses a [4Fe-4S] cluster of the site, and an FMN group (FMN-a) near the Ni-Fe site in the hoxH. It is also responsible for the role of conducting electrons between hoxH and hoxF.[4] The most important site in hoxH is the [NiFe] -hydrogenase active site, which is composed of Ni and Fe particles coordinated with cysteine residues, cyanide and carbon monoxide. [5] It is the most central component of our intracellular conversion of nickel ions. On its surface, oxidation and reduction of hydrogen gas happens alternately according to different oxygen status.
The rest of the subunits may work together to ensure that the hydrogenase can assemble and function well. It's worth noting that hypA and hypB can cooperate to precisely guide and insert the nickel ions into the hydrogenase catalytic center.[6]
Through the synergistic integration of the hox and hyp operon, our system effectively enhances hydrogen production and enables the reduction of nickel ions into nanoparticles, thereby maximizing the efficiency of nickel recovery from industrial wastewater.
Characterization
Agarose gel electrophoresis
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| Figure 1. Agarose gel electrophoresis of PCR products, amplified from bacterial colonies/cultures.
M: DNA Marker; Lanes 1-8: Amplification of specific regions corresponding to hoxF, hoxU, hoxI, hoxH, hoxW, hoxY, hypA, and hypB, indicating the correct assembly of the hydrogenase subunits derived from the target organism's genome. |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 485
Illegal BglII site found at 563
Illegal BglII site found at 9209
Illegal BglII site found at 10027
Illegal BglII site found at 10320
Illegal BamHI site found at 956
Illegal XhoI site found at 493
Illegal XhoI site found at 685
Illegal XhoI site found at 1163
Illegal XhoI site found at 3402
Illegal XhoI site found at 5231
Illegal XhoI site found at 6393
Illegal XhoI site found at 8232 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1256
Illegal NgoMIV site found at 1936
Illegal NgoMIV site found at 4543
Illegal NgoMIV site found at 4650
Illegal NgoMIV site found at 4920
Illegal NgoMIV site found at 5766
Illegal NgoMIV site found at 5998
Illegal AgeI site found at 446
Illegal AgeI site found at 5718
Illegal AgeI site found at 11103 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 267
Illegal BsaI site found at 429
Illegal BsaI site found at 3056
Illegal BsaI.rc site found at 9257
Illegal BsaI.rc site found at 9761
Illegal SapI.rc site found at 378
References
- ↑ Teramoto, H., Shimizu, T., Suda, M., & Inui, M. (2022). Hydrogen production based on the heterologous expression of NAD+-reducing [NiFe]-hydrogenase from Cupriavidus necator in different genetic backgrounds of Escherichia coli strains. International Journal of Hydrogen Energy, 47(52), 22010–22021.
- ↑ Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.
- ↑ Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.
- ↑ Löscher, S., Burgdorf, T., Zebger, I., Hildebrandt, P., Dau, H., Friedrich, B., & Haumann, M. (2006). Bias from H2 Cleavage to Production and Coordination Changes at the Ni−Fe Active Site in the NAD+-Reducing Hydrogenase from Ralstonia eutropha. Biochemistry, 45(38), 11658–11665.
- ↑ Chan, K.-H., Lee, K.-M., & Wong, K.-B. (2012). Interaction between Hydrogenase Maturation Factors HypA and HypB Is Required for [NiFe]-Hydrogenase Maturation. PLOS ONE, 7(2), e32592.
- ↑ Anne K. Jones, Oliver Lenz, Angelika Strack, Thorsten Buhrke, and, & Friedrich*, B. (2004, October 2). NiFe Hydrogenase Active Site Biosynthesis: Identification of Hyp Protein Complexes in Ralstonia eutropha† (world) [Research-article]. ACS Publications; American Chemical Society.
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