Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: iGEM11_UNAM-Genomics_ Mexico
Date created: 2011-08-23 11:00:00
Date modified: 2015-05-08 01:12:36
HydEF -> C. reinhardtii
| Types | DnaRegion |
| Roles | CDS Coding |
| Sequences | BBa_K535000_sequence (Version 1) |
Description
HydEF is a protein from Chlamydomonas reinhardtii genome, this protein contains two unique domains that are homologous to two distinct prokaryotic proteins, HydE and HydF, which are found exclusively in organisms containing [Fe] hydrogenase.In the C. reinhardtii genome, the HydEF gene is adjacent to another hydrogenase-related gene, HydG. All organisms with [Fe] hydrogenase and sequenced genomes contain homologues of HydE, HydF, and HydG.
HydEF and HydG genes are transcribed anaerobically in parallel with the HydA1 and HydA2 [Fe] hydrogenase genes in C. reinhardtii.
HydEF and HydG function in the assembly of [Fe] hydrogenase, they are accessory genes required for the maturation of an active [Fe] hydrogenase, thats why they are called "maturases".
The [Fe] hydrogenase (HydA1) catalytic site is known as the H-cluster and consists of a [4Fe4S] cluster connected through a bridging cysteinyl ligand to a binuclear [2Fe] center.
HydEF and HydG proteins belong to the Radical S-adenosylmethionine (designated "Radical SAM", also known as the AdoMet radical) superfamily because they contain the signature Cys-X3-Cys-X2-Cys motif that is typically found within the Radical SAM protein superfamily. This motive coordinates a redox active [4Fe4S] cluster under reducing conditions.
There is precedent for the involvement of a Radical SAM protein in the donation of iron to the catalytic metal cluster of an [Fe]-metalloenzyme, thats why its proposed that HydEF and/or HydG proteins play a similar role in the mobilization of iron for assembly of the [Fe] hydrogenase H-cluster.
The H-cluster also requires CN, CO, and the putative di(thiomethyl)amine ligand. It is conceivable that the accessory proteins HydEF and/or HydG are also responsible for the biosynthesis and assembly of these products coordinated to iron.
What differentiates HydEF from other proteins from the radical SAM superfamily are two conserved sequences with the potential to coordinate metal ions. These include a E(A/G)CXH and a (I/V)HC(G/A)(G/A)C motif near the C terminus of HydF, these motifs are strictly conserved in the [Fe] hydrogenase assembly proteins, but they are absent from other Radical SAM proteins.
The N-terminal portion of the HydEF protein is homologous to a group of proteins that, to date, are only found in prokaryotes containing [Fe] hydrogenases and belongs to a previously uncharacterized subset of the Radical SAM protein superfamily.
C-terminal portion of the HydEF protein contains a domain with predicted GTPase activity. This domain is homologous to a second distinct group of prokaryotic proteins, which are also unique to organisms that contain [Fe] hydrogenases
these proteins are required for the assembly of active [Fe] hydrogenase.
It has been demostrated that mutant C. reinhardtii strains with the HydEF gene inactivated are capable of prudicing [Fe] Hydrogenase but are incapable of produce H2, which indicates that the [Fe] Hydrogenase is not mature enough without this gene.
Notes
Some codons of the original Chalmidomonas' sequence had been changed for synonimous ones (according to the CAI procedure) in order to optimize its expression in Rhizobium etli.The sequence posses an unwanted insertion in the second spacer because of an in silico problem during its design.
Two TAA stop codons had been added at the end of the coding region.
Unwanted restriction sites had been changed for synonimous codons.
The constitutive promoter from LacZ has been added to regulate the expression of HydEF.
This sequence was synthesized.
Source
This sequence originaly comes from the genome of the unicelular photosynthetic green alga Chlamydomonas reinhardtii, but as specified in "sequence considerations" it has been modified in some parts.| Sequence Annotation | Location | Component / Role(s) |
| pLacZ TSS spacer RBS spacer HydEF double terminator insertion | 1,38 39,41 42,59 60,66 67,93 94,3546 3547,3552 73,93 | promoter feature/promoter feature/start start_codon feature/misc sequence_feature ribosome_entry_site feature/rbs sequence_feature feature/misc CDS feature/cds feature/stop stop_codon feature/misc sequence_feature |