Part:BBa_K2300000
HydEFG
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1849
Illegal NheI site found at 2059
Illegal NheI site found at 2503
Illegal NotI site found at 253 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 901
Illegal NgoMIV site found at 4857
Illegal AgeI site found at 1816
Illegal AgeI site found at 3459 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2684
Illegal BsaI.rc site found at 304
Illegal BsaI.rc site found at 416
Overview
HydEFG is a composite part which includes the maturation enzymes required to make a functional Hyd1 protein. The proteins in HydEFG (HydEF and HydG) are responsible for the insertion of the H-cluster in Hyd1 which is the catalytic core of the protein (Mulder et al., 2010).
All genes within this plasmid are sequences obtained from Chlamydomonas reinhardtii and codon optimised to be expressed in Escherichia coli.
Biology & Literature
In all [FeFe] hydrogenases, the H-cluster includes a di-iron subcluster that contains azadithiolate, three CO, and two CN− ligands (Dinis et al., 2015).
During the assembly of the H cluster, the HydG radical SAM enzyme lyses the substrate tyrosine to yield CO- and CN- ligands. These diatomic products serve as a precursor for eventual H-cluster assembly (Dinis et al., 2015).
HydG may also facilitate the transfer of the intact (CO)x(CN)y synthon to its cognate acceptor for the assembly of the H-cluster (Driesener et al., 2013).
HydEF in the majority of [FeFe] containing organisms exists as two separately transcribed proteins (HydE and HydF). However in several algal species the two genes are fused (Böck et al., 2006). HydE (along with HydG) is a radical SAM (S-adenosyl methionine) enzyme which binds [4Fe-4S] clusters in the biosynthesis of the H-cluster (Mulder et al., 2010). HydF contains an N-terminal GTPase domain which is responsible for the P loop motif and FeS binding (Mulder et al., 2010). Both these genes have been shown to be essential for the maturation of the HydA hydrogenase (King et al., 2006). This is due to their role in the creation of the H-cluster.
Part Verification
The entire Hydrogen Gas Producing Gene Cluster (BBa_K2300001) was sequenced and confirmed once it had been ligated together. This included the HydEFG part.
To confirm the efficacy of the ribosome binding sites in our parts we used the Salis Lab Ribosome Binding Site calculator from Penn State University. The results from this were that our ribosome binding site had a translation initiation rate of 1324.3.
Left: Lane 1 contains a 1kb ladder. Lanes 2 and 3 show single (~10,700bp) and double (~8700bp with ~2000bp) digests respectively of the composite Hydrogen Gas Producing Gene Cluster plasmid (HGPGC). Lanes 4 and 5 show single (~7400bp) and double (faint ~5400bp with ~2000bp) digests of hydEFG. Lanes 6 and 7 show single (~5400bp) and double digests (~3400bp with ~2000bp) of fer/hyd1.
Right: Lane 1 contains a 1kb ladder. Lanes 2 and 3 show double digests (~1900bp with ~2000bp) and single digest (~3900bp) of hydG.Protein information
HydEF
Mass: 121.95 kDa
Sequence:
MAHSLSAHSRQAGDRKLGAGAASSRPSCPSRRIVRVAAHASASKATPDVPVDDLPPAHARAAVAAANRRARAMASAEAAAETLGDFLGLGKGGLSP
GATANLDREQVLGVLEAVWRRGDLNLERALYSHANAVTNKYCGGGVYYRGLVEFSNICQNDCSYCGIRNNQKEVWRYTMPVEEVVEVAKWALENGI
RNIMLQGGELKTEQRLAYLEACVRAIREETTQLDLEMRARAASTTTAEAAASAQADAEAKRGEPELGVVVSLSVGELPMEQYERLFRAGARRYLIRIET
SNPDLYAALHPEPMSWHARVECLRNLKKAGYMLGTGVMVGLPGQTLHDLAGDVMFFRDIKADMIGMGPFITQPGTPATDKWTALYPNANKNSHMK
SMFDLTTAMNALVRITMGNVNISATTALQAIIPTGREIALERGANVVMPILTPTQYRESYQLYEGKPCITDTAVQCRRCLDMRLHSVGKTSAAGVWGDPA
SFLHPIVGVPVPHDLSSPALAAAASADFHEVGAGPWNPIRLERLVEVPDRYPDPDNHGRKKAGAGKGGKAHDSHDDGDHDDHHHHHGAAPAGAAA
GKGTGAAAIGGGAGASRQRVAGAAAASARLCAGARRAGRVVASPLRPAAACRGVAVKAAAAAAGEDAGAGTSGVGSNIVTSPGIASTTAHGVPRINI
GVFGVMNAGKSTLVNALAQQEACIVDSTPGTTADVKTVLLELHALGPAKLLDTAGLDEVGGLGDKKRRKALNTLKECDVAVLVVDTDTAAAAIKSGRLA
EALEWESKVMEQAHKYNVSPVLLLNVKSRGLPEAQAASMLEAVAGMLDPSKQIPRMSLDLASTPLHERSTITSAFVKEGAVRSSRYGAPLPGCLPRW
SLGRNARLLMVIPMDAETPGGRLLRPQAQVMEEAIRHWATVLSVRLDLDAARGKLGPEACEMERQRFDGVIAMMERNDGPTLVVTDSQAIDVVHPW
TLDRSSGRPLVPITTFSIAMAYQQNGGRLDPFVEGLEALETLQDGDRVLISEACNHNRITSACNDIGMVQIPNKLEAALGGKKLQIEHAFGREFPELESG
GMDGLKLAIHCGGCMIDAQKMQQRMKDLHEAGVPVTNYGVFFSWAAWPDALRRALEPWGVEPPVGTPATPAAAPATAASGV
HydG
Mass: 63.74 kDa
Sequence:
MSVPLQCNAGRLLAGQRPCGVRARLNRRVCVPVTAHGKASATREYAGDFLPGTTISHAWSVERETHHRYRNPAEWINEAA
IHKALETSKADAQDAGRVREILAKAKEKAFVTEHAPVNAESKSEFVQGLTLEECATLINVDSNNVELMNEIFDTALAIKE
RIYGNRVVLFAPLYIANHCMNTCTYCAFRSANKGMERSILTDDDLREEVAALQRQGHRRILALTGEHPKYTFDNFLHAVN
VIASVKTEPEGSIRRINVEIPPLSVSDMRRLKNTDSVGTFVLFQETYHRDTFKVMHPSGPKSDFDFRVLTQDRAMRAGLD
DVGIGALFGLYDYRYEVCAMLMHSEHLEREYNAGPHTISVPRMRPADGSELSIAPPYPVNDADFMKLVAVLRIAVPYTGM
ILSTRESPEMRSALLKCGMSQMSAGSRTDVGAYHKDHTLSTEANLSKLAGQFTLQDERPTNEIVKWLMEEGYVPSWCTAC
YRQGRTGEDFMNICKAGDIHDFCHPNSLLTLQEYLMDYADPDLRKKGEQVIAREMGPDASEPLSAQSRKRLERKMKQVLEGEHDVYL
References
Böck, A., King, P.W., Blokesch, M. and Posewitz, M.C., 2006. Maturation of hydrogenases. Advances in Microbial Physiology, 51, pp.1-225.
Dinis, P., Suess, D.L., Fox, S.J., Harmer, J.E., Driesener, R.C., De La Paz, L., Swartz, J.R., Essex, J.W., Britt, R.D. and Roach, P.L. 2015. X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly. Proceedings of the National Academy of Sciences, 112, 1362-1367.
Driesener, R.C., Duffus, B.R., Shepard, E.M., Bruzas, I.R., Duschene, K.S., Coleman, N.J.R., Marrison, A.P., Salvadori, E., Kay, C.W., Peters, J.W. and Broderick, J.B., 2013. Biochemical and kinetic characterization of radical S-adenosyl-L-methionine enzyme HydG. Biochemistry, 52, 8696-8707.
King, P.W., Posewitz, M.C., Ghirardi, M.L. and Seibert, M., 2006. Functional studies of [FeFe] hydrogenase maturation in an Escherichia coli biosynthetic system. Journal of Bacteriology, 188(6), pp.2163-2172.
Mulder, D.W., Boyd, E.S., Sarma, R., Lange, R.K., Endrizzi, J.A., Broderick, J.B. and Peters, J.W., 2010. Stepwise [FeFe]-hydrogenase H-cluster assembly revealed in the structure of HydA [Dgr] EFG. Nature, 465(7295), pp.248-251.
Mulder, D.W., Shepard, E.M., Meuser, J.E., Joshi, N., King, P.W., Posewitz, M.C., Broderick, J.B. and Peters, J.W., 2011. Insights into [FeFe]-hydrogenase structure, mechanism, and maturation. Structure, 19(8), pp.1038-1052.
Posewitz, M.C., King, P.W., Smolinski, S.L., Zhang, L., Seibert, M. and Ghirardi, M.L., 2004. Discovery of two novel radical S-adenosylmethionine proteins required for the assembly of an active [Fe] hydrogenase. Journal of Biological Chemistry, 279(24), pp.25711-25720.
Vancouver
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