Gene Report

Posted on March 10, 2022 by Cheapest Assignment

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Assessment Task - Video Essay

Identification of Protein the DNA sequence encodes

The allotted DNA sequence was BLAST on blastn suite present on NCBI, the search was carried out on default parameters. The result of BLASTn was shown in Figure.1. The gene encodes for cytochrome c3 in Desulfovibrio Vulgaris with Query coverage (QC) and Percentage Identity (PI) of 100%. Other genes predicted were of relatively less QC and PI values, hence, the gene under consideration was of cytochrome c3.

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For protein identification, the DNA sequence allotted was translated by Expasy Translate shown in Figure. 2. Output was then BLAST in blast suite. The results show that this sequence codes for Cytochrome c3 that is conserved in multispecies of genus Desulfovibrio by QC and PI of 100 percent as shown in Figure. 3. Other proteins that were also, found in the results although considering QC and PI as filters the best-suited protein was Cytochrome c3. Protein alignment with the query sequence was supplemented in Supplementary Figure. 1.

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Figure. 1: BLASTn result for the DNA sequence

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Figure. 2: DNA sequence translated by Expasy Translate for Protein BLAST

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Figure. 3: BlastP results for the sequence

Gene and Protein Characteristics of Cytochrome c3

Gene of cytochrome c3 in D. Vulgaris contain 674 nucleotides and GC content is 58.01% (Accession No. X04304) (https://www.ncbi.nlm.nih.gov/nuccore/X04304). The Source of DNA was from Desulfovibrio Vulgaris str. Hildenborough. (Voordouw and Brenner, 1986). In the GenBank file, they reported a signal peptide at position 153-218 nucleotide position. CDS is in position 153-542 nucleotide sequence that constitute 57.8% of the whole gene length.

The protein sequence of Cytochrome c3 in D. Vulgaris contains 129 amino acids with a molecular size of 13.98 kDa (Accession No. WP_010940429.1) (https://www.ncbi.nlm.nih.gov/protein/40821). The protein sequence contains 1 to 129 residues (Voordouw and Brenner, 1986). GenBank file showed the presence of signal peptide at 1-22 position while 23-128 comprise of protein and E residue is an additional amino acid present in the structure that is not involved in the overall protein functioning. (Voordouw and Brenner, 1986).

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Introduction to Cytochrome c3 in D. Vulgaris

Dense packing of heme groups of cytochrome c3 was ensured by covalent heme attachment. Bacterial cytochrome c3 are either localized on either periplasm or at the outer membrane of Gram-negative bacteria. Cytochrome c3 in prokaryotes usually function in anaerobic respiration, specifically electron transport chains and the growth of these anaerobic microorganisms usually depends on one of the different types of these c-type cytochromes.

All cytochromes c3 contain eight residues of cysteine, that connect pairwise to four c-type heme groups by thioether bridges. Cytochrome c3 in D. Vulgaris consist of metalloproteins that can perform various biochemical functions including electron transport chains and enzymatic activity. (Sharma, Cavallaro and Rosato, 2010). Cytochrome c3 are usually present in sulfate-reducing organisms (SRO) like D. Vulgaris that are linked with diverse respiratory pathways involved in the utilization of elemental sulfur and act as a terminal electron acceptor. (Fauque et al., 1988; Le Faou et al., 1990; Matias et al., 2005; Fauque and Barton, 2012). Cytochrome class 3 (c3) are classified according to Ambler (1991) due to the presence of bis-histidine axial coordination and low redox potential. Other members of this class include tri-hemic, tetra hemic, octa-hemic, and nine-hemic cytochromes c. Cytochrome c3 belonging to this class III is characterized by the presence of four hemes in a low-spin state with bis- histidine coordination and quite negative redox potentials. Cytochrome c3 is the only hemoprotein reported only in this species. (Le Faou et al., 1990; Matias et al., 2005; Fauque and Barton, 2012).

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Cytochrome c3 is a small monomeric hemoprotein located in the periplasmic space. Cytochrome c3 plays a major role in bioenergetics of sulfate reduction, which mediated the flow of electrons from membrane/periplasmic hydrogenases to electron transport complexes (that are involved in respiration) coupled to the transfer of protons. (Matias et al., 2005; Pereira, Linden and Weinberg, 2007).

Cytochrome c3 are also involved in the metal reduction in an anaerobic environment hence It’s role is crucial in the survival of microorganism, In a review by (Fauque and Barton, 2012)  showed that D. Vulgaris can reduce Sulphur, Fe (III), Cr (VI), Mn (IV), U(VI) and Se (VI). This reductase activity is actually due to the activity of cytochrome c3 in combination with other survival cycles. (Fauque and Barton, 2012). Reduction of U(VI) was well studied in D. Vulgaris as it is used as a model organism for the Sulphur and uranium reduction model. This Uranium reduction function was experimentally proved when the hydrogenase alone was used with cytochrome c3 extracted and purified by cation column, D. Vulgaris lost its ability to reduce Uranium. Although with the addition of cytochrome c3, it regained its ability. It was a long thought before that D. Vulgaris only reduces Sulphur but now it reduces more Uranium than Sulphur in a solution media. (Elias et al., 2004). Cytochrome c3 reduces Uranium in a solution and converts soluble U (VI) to insoluble U(VI) in a solution. Cytochrome c3 works coupled with hydrogenase that accepts the electrons of Uranium and hence the Uranium can be cleared. This mechanism was widely used for the biostimulation and bioremediation on site for the clearance of various metals, Uranium and sulphur. (Lovley et al., 1993). Previously, it was also thought that cytochrome c contains the binding site of heavy metals but it is now seen that only specific metals can bind with the cytochrome c and hence can be reduced by other underlying mechanisms in the microbial cell. The only problem with metal reduction with Mn (VI) or U (VI) is that D. Vulgaris do not use these metals as the final electron acceptor in the electron transport chain or in respiration hence, growth is the main underlying factor concerned of this microorganism. (Le Faou et al., 1990; Lovley et al., 1993; Lovley and Phillips, 1994; Fauque and Barton, 2012; Franco et al., 2018). To combat that and to use it for the metal reduction and in broader terms for bioremediation, supplements of carbon, nitrates and Sulphur are added that increase the growth of microorganisms that drastically reduce the contaminants at a site (Mn or U).

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Post Translation Modification and Localization of Cytochrome c3

The sequence of cytochrome c3 in D. Vulgaris is composed of 22 amino acid long signal peptides and the remaining 106 amino acid long chain. Signal peptides direct their export to the endoplasmic reticulum after translation by ribosomes. The endoplasmic reticulum pack the protein properly and directs it to the periplasm or to the outer membrane, where the signal peptides were chopped by the help of peptidases. Cytochrome c3 forms an electron transport channel there, that with the help of membrane hydrogenases help in the survival of anaerobically respiring gram-negative bacteria.

3D structure of Cytochrome c3 in D. Vulgaris (1A2I)

The 3D structure was checked by BLAST sequence in the Uniprot database. The 3D structure of Cytochrome c3 has PDB entry 1A2I (https://www.uniprot.org/uniprot/P00131#structure) was NMR based structure published in 1998. The 3D structure revealed that the protein contains 8 Beta strands and 5 Alpha Helix and 1 turn. (Valente et al., 2001).

Overall, it is a soluble protein crystallized structure that was determined previously by X-ray diffraction in the oxidized state but NMR structure solved by experimental evidence. (Messias et al., 1998). The 3D structure also revealed the presence of the Tetra-heam group in the protein structure. (Matias et al., 2005). Heam groups form a complex network of centres that have redox potential. Hence, resulting in homotropic or redox cooperativity between their redox potentials. (Turner et al., 1996; Matias et al., 2005). This cooperativity is also dependent on the pH of the solution, inferring heterotopy cooperativity between both their heam redox and protonation energies. (Moura et al., 1982; Turner et al., 1996; Messias et al., 1998). Overall, these properties jointly give the ability to protein to transfer two electrons and two protons in the joint step. This electron transport when paired with the oxidation of molecular hydrogen catalysed by hydrogenase results in a transition from electronic to protonic energy achieved in the absence of membrane confinement usually referred to as proton thrusters. (Louro et al., 1996, 1997).

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Structural studies revealed that in all four heme, there are two Histidines that act as ligands to the 5th and 6th position of coordination of the central iron atom, contrasting to the coordination of the single c-type heme in eukaryotic cytochrome c by a histidine and a methionine residue. All four hemes situated in different environments do not have the same redox potential. (Messias et al., 1998). The relative position of the hemes is identical to D. desulfuricans Norway and D. Vulgaris Miyazaki. Cytochrome c3 of D. Vulgaris (Hildenborough) similarity to cytochrome c3 of D. desulfuricans Norway by 30% and is 90% similar to cytochrome c3 of D. Vulgaris Miyazaki. As the hydrogenase can directly react with cytochrome c3 in-vitro, there is a strong implication that they are linked in-vivo. (Voordouw and Brenner, 1986; Messias et al., 1998).

3D Structure of Cytochrome c3 of D. Vulgaris is shown in the Figure. 4. The details of the beta strand and alpha helixes in the structure is shown in Table. 1.

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Figure. 4: 3D structure (NMR) of Cytochrome c3 protein of D. Vulgaris

Table. 1: Beta strands, alpha-helix and turns in the 3D structure of Cytochrome c3 of D. Vulgaris

Feature key Position(s) Amino Acid Length
Beta strand 31 – 33 3
Beta strand 35 – 38 4
Beta strand 40 – 42 3
Helix 45 – 47 3
Beta strand 48 – 50 3
Helix 52 – 54 3
Beta strand 60 – 63 4
Turn 70 – 72 3
Beta strand 83 – 85 3
Helix 87 – 92 6
Beta strand 95 – 98 4
Helix 101 – 109 9
Helix 113 – 120 8
Beta strand 122 – 127 6

Role of Cytochrome c3 at Molecular Level in D. Vulgaris

Uniprot gives information about the molecular function of any protein with the help of Gene Ontology (GO) databases. (Ashburner et al., 2000; Ashburner et al., 2019). Data at GO databases are curated and linked by different other databases like Pfam, ProCite and InterPro. Cytochrome c3, as predicted by GO biological function and biological functions shows that it is involved in electron transfer activity, heme binding and metal ion binding at a molecular level in D. Vulgaris. Overall, the molecular function is that it participates in sulfate respiration coupled with phosphorylation by transferring electrons from the enzyme dehydrogenase to ferredoxin (Voordouw and Brenner, 1986).

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Desulfovibrio species are model sulfate-reducing bacteria (SRB) and they have the ability to reduce metals, metalloids, and radionuclides. (Lovley and Phillips, 1994; Heidelberg et al., 2004) It has been shown in previous studies that D. Vulgaris can reduce Cr (VI) by the help of hydrogenase and cytochrome c3. Cells, however, are not able to use Cr(VI) as the terminal electron acceptor that can be linked to the growth of microorganisms. (Lovley and Phillips, 1994; Franco et al., 2018)

Pfam Analysis of Cytochrome c3 of D. Vulgaris

The protein sequence of Cytochrome c3 of D. Vulgaris was retrieved from NCBI (Accession No. WP_010940429.1) and copied to Notepad++ for the creation of fasta file. The protein fasta file was then searched on the Pfam database of EMBL-EBI (http://pfam.xfam.org/). The result revealed that our protein belongs to Family: Cytochrom_CIII (PF02085). Domain organization revealed that domains of this protein are present in 27 different domain organizations ranging from 1-361 sequences present in different organisms. Some species were having the same genus however species like Saprospira Grandis also share domain in the sequence. (http://pfam.xfam.org/family/PF02085.16#tabview=tab1).

Our protein also contains interactions of domains at 3D structure and maps level that showed interaction with three different domains; 4Fe-4S binding domain, Cytochrome c3 and Iron hydrogenase small subunit that refers about its function as Iron binding, electron transport and hydrogenase respectively.

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Prostate Analysis of Cytochrome c3 of D. Vulgaris

Protein Fasta sequence of Cytochrome c3 retrieved from NCBI was searched on Prosite by Extasy (https://prosite.expasy.org/) for finding the functional sites and protein family data that are present on  Prosite database. ScanProsite revealed the presence of multiheme cytochrome c motif present at 44-110 amino acid site, N-glycosylation site at 43-46 amino acid, N-myristoylation site present at 110-115 and 121-126 site. Results can be visualized in Figures 5 and 6.

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Figure. 5: Output of ScanProsite showing the Multiheme motif present in Cytochrome c3 of D. Vulgaris

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Figure. 6: Output of ScanProsite showing motifs present in Cytochrome c3 of D. Vulgaris

Conclusion

DNA sequence provided by Dr Paul Curley was of cytochrome c3 gene of D. Vulgaris. The 3D structure was present in Uniprot and was well studied and supported by experimental analysis. Cytochrome c3 in D. Vulgaris has functions of electron transport, reduction, and binding with metals. These functions of cytochrome c3 are crucial for the survival of anaerobically respiring gram-negative microorganisms. Multiheme and overall protein structure are conserved in multiple species of genus Desulphovibrio. Different motifs were also present in cytochrome c3 beside multiheme group that helps in binding and reduction of metals and elements like Uranium. Hence, it can be used for bioremediation and biostimulation.

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References

Ashburner, M. et al. (2000) ‘Gene ontology: a tool for the unification of biology. The Gene Ontology Consortium.’, Nature genetics, 25(1), pp. 25–29. DOI: 10.1038/75556.

Elias, D. A. et al. (2004) ‘Periplasmic Cytochrome <em>c</em><sub>3</sub> of <em>Desulfovibrio vulgaris</em> Is Directly Involved in H<sub>2</sub>-Mediated Metal but Not Sulfate Reduction’, Applied and Environmental Microbiology, 70(1), pp. 413 LP – 420. doi: 10.1128/AEM.70.1.413-420.2004.

Le Faou, A. et al. (1990) ‘Thiosulfate, polythionates and elemental sulfur assimilation and reduction in the bacterial world’, FEMS Microbiology Reviews. Blackwell Publishing Ltd Oxford, UK, 6(4), pp. 351–381.

Fauque, G. et al. (1988) ‘The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio’, FEMS microbiology reviews. Blackwell Publishing Ltd Oxford, UK, 4(4), pp. 299–344.

Fauque, G. D. and Barton, L. L. (2012) ‘Chapter 1 – Hemoproteins in Dissimilatory Sulfate- and Sulfur-Reducing Prokaryotes’, in Poole, R. K. (ed.). Academic Press (Advances in Microbial Physiology), pp. 1–90. doi: https://doi.org/10.1016/B978-0-12-398264-3.00001-2.

Franco, L. C. et al. (2018) ‘Cr(VI) reduction and physiological toxicity are impacted by resource ratio in Desulfovibrio Vulgaris, Applied Microbiology and Biotechnology, 102(6), pp. 2839–2850. doi: 10.1007/s00253-017-8724-4.

Heidelberg, J. F. et al. (2004) ‘The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio Vulgaris Hildenborough’, Nature biotechnology. Nature Publishing Group, 22(5), pp. 554–559.

Louro, R. O. et al. (1996) ‘Redox-Bohr effect in the tetrahaem cytochrome c 3 from Desulfovibrio vulgaris: a model for energy transduction mechanisms’, JBIC Journal of Biological Inorganic Chemistry. Springer, 1(1), pp. 34–38.

Louro, R. O. et al. (1997) ‘Redox-Bohr effect in electron/proton energy transduction: cytochrome c 3 coupled to hydrogenase works as a proton thruster’s Desulfovibrio Vulgaris, JBIC Journal of Biological Inorganic Chemistry. Springer, 2(4), pp. 488–491.

Lovley, D. R. et al. (1993) ‘Reduction of uranium by cytochrome c3 of Desulfovibrio Vulgaris, Applied and environmental microbiology, 59(11), p. 3572—3576. DOI: 10.1128/aem.59.11.3572-3576.1993.

Lovley, D. R. and Phillips, E. J. P. (1994) ‘Reduction of chromate by Desulfovibrio Vulgaris and its c3 cytochrome’, Applied and Environmental Microbiology. Am Soc Microbiol, 60(2), pp. 726–728.

Matias, P. M. et al. (2005) ‘Sulphate respiration from hydrogen in Desulfovibrio bacteria: a structural biology overview’, Progress in biophysics and molecular biology. Elsevier, 89(3), pp. 292–329.

Messias, A. C. et al. (1998) ‘Solution structure of Desulfovibrio Vulgaris (Hildenborough) ferrocytochrome c3: structural basis for functional cooperativity11Edited by P. E. Wright’, Journal of Molecular Biology, 281(4), pp. 719–739. DOI: https://doi.org/10.1006/jmbi.1998.1974.

Moura, J. J. G. et al. (1982) ‘Unambiguous identification of the nickel EPR signal in 61Ni-enriched Desulfovibrio Gigas hydrogenase’, Biochemical and biophysical research communications. Elsevier, 108(4), pp. 1388–1393.

Pereira, V. J., Linden, K. G. and Weinberg, H. S. (2007) ‘Evaluation of UV irradiation for photolytic and oxidative degradation of pharmaceutical compounds in water, Water Research. Elsevier, 41(19), pp. 4413–4423.

Sharma, S., Cavallaro, G. and Rosato, A. (2010) ‘A systematic investigation of multiheme c-type cytochromes in prokaryotes’, JBIC Journal of Biological Inorganic Chemistry. Springer, 15(4), pp. 559–571.

‘The Gene Ontology Resource: 20 years and still GOing strong.’ (2019) Nucleic acids research, 47(D1), pp. D330–D338. DOI: 10.1093/var/gky1055.

Turner, D. L. et al. (1996) ‘NMR studies of cooperativity in the tetrahaem cytochrome c3 from Desulfovibrio Vulgaris, European journal of biochemistry. Wiley Online Library, 241(3), pp. 723–731.

Valente, F. M. et al. (2001) ‘A membrane-bound cytochrome c3: a type II cytochrome c3 from Desulfovibrio Vulgaris  Hildenborough.’, Chembiochem : a European journal of chemical biology. Germany, 2(12), pp. 895–905. DOI: 10.1002/1439-7633(20011203)2:12<895::AID-CBIC895>3.0.CO;2-V.

Voordouw, G. and Brenner, S. (1986) ‘Cloning and sequencing of the gene encoding cytochrome c3 from Desulfovibrio Vulgaris (Hildenborough).’, European journal of biochemistry. England, 159(2), pp. 347–351. DOI: 10.1111/j.1432-1033.1986.tb09874.x. 

Supplementary Material

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  1. Figure. 1: Alignment of Query/Allotted sequence with Cytochrome c3 (Result of BlastP)
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