IGEM:UNAM-Genomics Mexico/2009/Notebook/H2/2011/03/21

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Assembly Process

 * 1) HydA - [4Fe4S] cluster loading by host machinery
 * 2) HydF - GTP hydrolisis to adopt scaffold conformation
 * 3) HydG - SAM activity synthesizes CN & CO from Tyrosine to stabilize secondary [FeFe] cluster
 * 4) HydE - SAM activity synthesizes dithiolate from Cysteine to stabilize mature [FeFe] cluster
 * 5) HydF - secondary cluster insertion into HydA once it is matured
 * 6) HydA - [4Fe4S] cluster with [FeFe] cluster on HydA constitute the mighty H cluster
 * "The structure indicates that H-cluster synthesis occurs in a stepwise manner, first with synthesis and insertion of the [4Fe-4S] subcluster by generalized host-cell machinery and then with synthesis and insertion of the 2Fe subcluster by specialized hydE-, hydF- and hydG-encoded maturation machinery."
 * Source doi: 10.1038/nature08993

Consants

 * For the simpler SimBiology model, I've found the following constants:
 * HydF GTPase Kcat= 0.04 min−1 = 6.66E-4 sec-1
 * The Kcat value taken is in the relative inhibitory presence of NH4+ (similar to inhibition by K+).
 * Source doi:10.1073/pnas.1001937107
 * It would appear GTP hydorlysis is required for interaction with HydE & HydF by stabilization of a difficult conformation. Therefore it is required for the [FeFe] auxiliary cluster constitution.
 * Source doi:10.1002/anie.200907047
 * HydE SAM rate of "1 mol AdoH/mol protein/h", therefore a Kcat=2.7E-4 sec-1
 * SAM activity appears to be required to mature the H cluster by synthesis of the remaining non-protein ligand: dithiolate, "They [HydE & HydG] could convert dimethylamine (or propane) into the bridging compound through activation of the carbon atoms of the terminal methyl groups by the Radical-SAM cluster and the insertion of sulfur atoms from the additional clusters that are bound to the proteins." See note about Mystery AA
 * Source doi:10.1016/j.febslet.2005.07.092
 * HydG "turnover number for HydG (Table 1, kcat.0=(20±2)×10−4 s−1)", therefore Kcat=20E-4 sec-1
 * SAM activity of HydG appears to cleave tyrosine into cyanide and carbon monoxide, the two ligands required in the auxiliary [FeFe] cluster: "we have demonstrated that HydG catalyzes radical AdoMet chemistry, cleaving tyrosine to form a 1:1 stoichiometric ratio of cyanide to p-cresol." & "...a decarbonylation mechanism, which has chemical precedent, would yield both the cyanide and carbon monoxide ligands in a single step (NHCHCO2H→HCN+CO+H2O)."
 * Source dOI:10.1002/anie.200907047
 * HydA "Based on the known activity of 304 μmol H2 min−1 mg−1 for native [FeFe]-hydrogenase", therefore Kcat= ?
 * Source doi:10.1016/j.febslet.2008.04.063
 * HydA "726 μmol H2 min−1 mg protein−1"
 * Source doi:10.1016/j.febslet.2009.12.016
 * HydF maturation rate of HydA, I'll take 15% based on Intrinsic Maturases Issues note.
 * As for HydA activity, we have some interesting data: "The purified algal hydrogenases evolve hydrogen with rates of around 700 µmol H2 min–1 mg–1, while HydA from C. acetobutylicum (HydACa) shows the highest activity (5,522 µmol H2 min–1 mg–1) in the direction of hydrogen uptake". Therefore, which one will we take?
 * Source doi:10.1128/AEM.71.5.2777-2781.2005

Mystery AA

 * It would appear that in addition to tyrosine, cysteine is also a substrate for HydA maturation.
 * Source doi:10.1371/journal.pone.0007565

Intrinsic Maturases Issues

 * It appears that the intrinsic maturases found in our HydX donors help fold correctly our HydA maturases (HydE, HydF, HydG). In the abscence of this intrinsic maturases (that is, expressed in another host), the activity of HydF collapses dramatically, requiring an 80 fold overexpression to rescue levels to 15% or previous activity: "Interestingly, an about 80-fold excess of the HydF protein is needed to yield an H2 evolution rate of only 50 μmol H2 min−1 mg−1 (about 15% of the native activity) if HydF is heterologously expressed with a background of the other heterologously introduced maturation factors in E. coli ".
 * Source doi:10.1016/j.febslet.2009.12.016


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