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
PFOR -> D. africanus Pyruvate-Ferredoxin OxidoReductase
| Types | DnaRegion |
| Roles | CDS Coding |
| Sequences | BBa_K535006_sequence (Version 1) |
Description
Pyruvate-ferredoxin oxidoreductases (PFORs) are a widespread group of proteins from several anaerobic or microaerobic species of microorganisms, with a variety of functions in energy metabolism, and CO2?? fixation.Though they generally catalyze the crucial energy-yielding reaction of pyruvate oxidation and serve as electron carriers, sometimes they differ in distinct species in their acceptor/donor specificity, substrate specificity, sensitivity to O2, and kinetics of CO2 production and consumption.
They are complex iron-sulfur proteins containing thiamine pyrophosphate (TPP) and one or two iron-sulfur [4Fe-4S] clusters. They contain neither flavin nor lipoate as prosthetic groups.
This sequence corresponds to the PFOR from Desulfovibrio africanus which is a Gram negative sulfate-reducing bacterium, mostly inhabiting anaerobic environments. PFOR in this organism has been reported to be located in the cytoplasm, like the reductases of the sulfate reduction pathway.
The specific activity of D. africanus PFOR is exceptionally high compared with other mesophile PFORs reported.
D. africanus PFOR is a 256??10 kDa homodimer which contains two thiamine pyrophosphate (TPP). Each subunit has three [4Fe-4S] 2+/1+ centers, which is higher than other PFORs which have at most two [4Fe-4S] clusters per subunit. The midpoint potentials of the three centers are -390 mV, -515 mV and -540 mV.
The three [4Fe-4S] clusters are close enough in the PFOR subunit to interact magnetically, namely less than about 1.5 nm apart.
In Desulfovibrio species the enzyme is involved in the oxidation of pyruvate and provides the sulfate reducing pathway with low potential electrons. Generally, ferredoxin and flavodoxin serve as electron acceptors for the enzyme. Ferredoxins are the most effective acceptor for the D. africanus enzyme.
In order to work, the enzyme should be activated by dithioerythritol. Neither the nature, nor the arrangement of [4Fe-4S] centers are affected by the activation process, nevertheless there is a change in the protein's conformation during activation which makes the enzyme very sensitive to oxygen in contrast to the enzyme before activation. This is believed to happen given that iron-sulfur clusters are now exposed to oxidative damage.
In our project we intend to create a pathway with heterologous expressed PFOR-ferredoxin-hydrogenase in order to couple the hydrogen production to the glycolisis process.
Notes
Codons of the original Desulfovibrio africanus sequence were changed for synonimous ones according to the Codon adaptation index (CAI) procedure in order to optimize its expression and Rhizobium etli???s fitness as well (where we will express the gene).The Codon Adaptation Index indicates how similar the Codon Usage (CU) in a coding sequence (CDS) is to those of highly or 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 being the most frequent codon, 0 being a non-existent codon) in R. etli and then we substituted codons in the target CDS with all synonymous codons with greatest w.
5' UTR and the first seven codons were optimized for Gibbs free energy to avoid formation of secondary and tertiary structures. This was achieved by taking ???allowed??? synonym codons (as determined by w higher than threshold) and using the combination of the first seven codons that together with the six nucleotide long sequence (generated using RSATools) as a spacer, had the least negative ∆G value which indicates a worse folding, relating to a better translation initiation in mRNA. We avoided sequences with restriction sites.
Two TAA stop codons had been added at the end of the coding region.
Pfor contains a periplasm export tag between two NdeI restriction sites intended for removal
Some standard restrictions sites arose from this optimization. They were removed by changing the codons spanning these sites for synonimous codons.
This sequence was synthesized.
Source
The coding sequence is part of the Desulfovibrio africanus genome. The 5??? UTR was designed by us to include a RBS and spacer sequences for translational optimization.| Sequence Annotation | Location | Component / Role(s) |
| spacer RBS spacer Periplasm export PFOR poly His tag double TAA stop | 1,15 16,22 23,28 29,166 167,3862 3863,3892 3893,3898 | sequence_feature feature/misc ribosome_entry_site feature/rbs sequence_feature feature/misc tag feature/tag feature/cds CDS tag feature/tag stop_codon feature/stop |