IGEM:UNAM-Genomics Mexico/2009/Notebook/H2/2011/03/18

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Ferredoxin Issues

  • Part1
    • For our FeFe hydrogenase, "Lower catalytic rates when using a [2Fe–2S] ferredoxin have been previously observed when compared to rates observed when a [4Fe–4S] ferredoxin was used [30]." From doi:10.1371/journal.pone.0015491
    • Sinorhizobium meliloti appears to have a related ferredoxin [MAFKITASQCTQCGACEFECPRGAVNFKGEKYVIDPTKCNECKXXXXXXQCASVCPVSNTXXXX]. From Fig. 1, DOI: 10.1021/bi00051a028
    • Blast analysis of the Sinorhizobium meliloti apparent ferredoxin yielded related proteins with E-value of around 10^(-17) in Rhizobium etli. Considering the reference is of a [4Fe-4S] ferredoxin, and many of the other ferredoxins from related organisms are also of [4Fe-4S] ferredoxins, I feel confident R. etli also has a [4Fe-4S] ferredoxin. The alignment is File:Hmedina S7700R3K01R-Alignment-HitTable.csv.
    • However, a search for conserved domains revealed possible similarities to "PRK09326: F420H2 dehydrogenase subunit F; Provisional [181779]". I interpret this as a possible association of the [4Fe-4S] clusters...
  • Part2
    • Moreover, when considering the C. acetobutylicum, C. saccharobutylicum, C. reinhardtii, & Shewanella oneidensis, "all hydrogenases produced the highest output when co-expressed with bacterial type 2-[4Fe-4S] ferredoxin from Clostridium acetobutylicum, with a potential of -420mV". From doi:10.1186/1754-1611-4-3
    • In fact, the greatest result came from chimaeric proteins: "The behavior of the fusion protein is similar with the ferredoxin from C. acetobutylicum, although the bacterial ferredoxin is equally active when fused at either terminus (figure 5C)." From the image, it appears a 14 AA linker is optimal. As for the composition, the paper strategy was "direct fusion by a flexible glycine/serine-rich linker". From doi:10.1186/1754-1611-4-3
    • Now the question is, can Phaseolum vulgaris give glucose to Rhizobium etli? If it does, we can insert the chimaeric protein plus the PFOR and obtain greater yields!

Maturases Issues

  • The modeling of the system is Simbiology will remain the simplified version for the moment. However, a Kappa model will be built to facilitate the explanation of the system. The (doi: 10.1073/pnas.1001937107) paper is quite explanatory on the role of HydF & the [Fe4S4] core.