Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: iGEM11_UNAM-Genomics_ Mexico
Date created: 2011-09-24 11:00:00
Date modified: 2015-05-08 01:12:36
HydA (hydrogenase I)-> Clostridium acetobutylicum ATCC 824
| Types | DnaRegion |
| Roles | Coding CDS |
| Sequences | BBa_K535002_sequence (Version 1) |
Description
Hydrogenases are a class of metalloenzymes that catalyze the reversible reduction of protons to molecular hydrogen 2H+ + 2e− ↔ H2 with an equilibrium constant that is dependent on the reducing potential of electrons carried by their redox partner.These metalloenzymes (containing metallo-catalytic clusters) are subdivided into two classes depending of the two metal atoms that are present at their active center: either a Ni and a Fe atom in the [NiFe]hydrogenases, or two Fe atoms in the [FeFe]hydrogenases. These two forms are phylogenetically distinct, which suggests that hydrogenase function is the result of convergent evolution.
[NiFe] hydrogenases are found across a variety of organisms, [FeFe] hydrogenases are typically restricted to algal species and to a few anaerobic prokaryotes, such as clostridia and sulfate reducers, but are excluded from all cyanobacteria examined to date.
Although [NiFe] and [Fe] hydrogenases are genetically unrelated, similarities between the proteins do exist. First, the active sites of both enzymes contain CO and CN ligands, and, second, each active site contains a binuclear metal center.
The hydrogenase presented here is a [FeFe] hydrogenase from Clostridium acetobutylicum ATCC 824 which is a Gram-positive bacteria that belongs to the Firmicutes division and that is an obligate anaerobe capable of produce endospores.
We choose this hydrogenase because [FeFe]-hydrogenases, such as those from Clostridium species contain several ???ferredoxin-like??? domains. It is speculated that these domains arose through ancestral gene fusions, enhancing hydrogenase interaction with other ferredoxins, and providing an electron transport channel towards the hydrogenase active site.
Because ferredoxin proteins may carry electrons with reducing potentials closer to that of the H2∕H+ pair (−420 mV), [FeFe]-hydrogenases thermodynamically favor hydrogen production relative to [NiFe] hydrogenases, which are generally coupled to NAD(P)H, with a reducing potential of -320 mV and are frequently regarded as predominantly H2 uptake enzymes.
[Fe] hydrogenase 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. In addition to binding CO and CN, the iron atoms of the [2Fe] center coordinate a bridging organic group thought to be a di(thio-methyl)amine moiety. The H-clusters of [Fe] hydrogenases are easily oxidized and are located in the interior of the protein structure. These sites are connected to the surface by a hydrophobic channel that facilitates H2 diffusion.
Because of the complexity of the [Fe] hydrogenase H-cluster assembly, the active hydrogenase expression require at least three accessory proteins called the HydE, HydF, and HydG maturases, like the [FeFe] hydrogenases, HydE, HydF, and HydG also require ISCs (iron-sulfur clusters).
The reduced nature of the H-cluster and accessory iron-sulfur clusters (ISCs) makes the Hydrogenase and the maturases susceptible to damage by O2 oxidation.
Notes
Some codons of the original Clostridium acetobutylicum ATCC 824 hydA sequence have been changed for synonimous ones according to the Codon Adaptation Index (CAI) procedure with respect to Rhizobium etli CFN42 codon usage in order to optimize its expression and to optimize R. etli CFN42???s (where we will express this gene) fitness as well.The Codon adaptation Index indicates how similar the Codon Usage (CU) in a coding sequence (CDS) is to that of highly/constitutively expressed genes. It is not a cause of high gene expression, but it is necessary to optimize resource usage. To optimize a sequence according to the CAI procedure we first obtained relative adaptiveness (w) for each codon (1.- most frequent codon. 0.- non-existent codon) in R. etli and then we substitute codons in target CDS with all synonymous codons with greatest w.
The sequence that we use is linked to the FeOx gene, at the end it has two TAA stop codons. Please refer to the composite part BBa_K535###.
Unwanted restriction sites had been changed for synonimous codons.
A NifH promoter from Rhizobium etli CFN42 has been added to regulate the expression of this construction because we want it to be expressed under the same conditions that the genes related to nitrogen fixation are expressed.
This sequence was synthesized .
Source
This sequence belongs to the Clostridium acetobutylicum ATCC 824 hydA hydrogenase gene which is in the chromosome of this organism. It has been modified, please refer to the ???design notes??? section for more details.| Sequence Annotation | Location | Component / Role(s) |
| Periplasm export tag HydA | 1,138 139,1884 | feature/tag tag CDS feature/cds |