Understanding the fluorescent system in a Prokaryotic and Eukaryotic organism by sequential and structural comparison of proteins involved in bioluminescence. Jerry.A. JOHN Post Graduate Department of Biotechnology, St. Xavier’s College (Autonomous),Mumbsi
Emission of light due to the interaction of Luciferase and Luciferin is called bioluminescence. The illuminating mechanism is different for Eukaryotic and Prokaryotic organisms. In the current study, the primary, secondary and tertiary characteristics of the Luciferase protein from Eukaryotic and Prokaryotic organisms were analyzed. The Bacterial Luciferase has a different gene associated with the mechanism whereas the Firefly Luciferase is an ATP dependent protein. Bacterial Luciferase has a heterodimeric structure as 2 genes i.e. Lux A and Lux B plays an important role in the emission of light in bacteria whereas Firefly Luciferase has a homodimeric structure. Sequentially Firefly Luciferase shares approximately 30% of identity with Bacterial Luciferase and structurally it seems to be quite different as little variation was analyzed in their Physio-chemical properties.
Certain prokaryotic and eukaryotic organisms exhibit phenomena of bioluminescence wherein they emit light naturally.A group of an enzyme known as Photoproteins which exhibit the property of bioluminescence by an enzymecatalyzed oxidation reaction in cells. In the case of prokaryotic bacterial systems, Flavin Monooxygenase Luciferase catalyzes long-chain aldehydes and releases energy in the form of fluorescent/visible light. This mechanism also occurs in Vibrio harveyi bacteria, wherein it is controlled by lux operon genes.
It is intriguing how prokaryotic and eukaryotic bioluminescent systems with different sets of genes have the same function. Thus, current projects focus on understanding similarities and differences between components of two systems at the sequence and structure level. To study both Luciferin 4-monooxygenase and Alkanal Monooxygenase â?ş -đ?›ƒ chain sequentially by using Jalview to identify their different conserved characteristics and by USCF Chimera to visualize their structural identity and molecular interaction.
Analyzing components of the bioluminescence system in prokaryotic and eukaryotic by sequence and structure comparison.
Retrieving ALKANAL MONOOXYGENASE ι-β CHAIN (P07740,P07739) & LUCIFERASE 4-MONOOXYGENASE (Q26304) protein sequence using UNIPORT
ď ą Primary characteristics 1.Physico-chemical properties: The total no. of negatively charged residues are more in all proteins as compared to total no. of positively charged residues. 2.Sequence Similarity: Luciferin 4-monooxygenase shares 35.71% of sequence similarity with Alkanal monooxygenase alpha chain while Alkanal monooxygenase beta chain shares only 27.03% of sequence similarity with deletion of 4 amino acid residues from Luciferin 4 monooxygenase . 3. MSA: The amino acid residues conserved across Luciferin 4monooxygenase and Alkanal monooxygenase alpha-beta chain were found to be Glutamic acid, Phenylalanine, Aspartic acid, Proline, Alaine, Glycine, Leucine, Valine, Methionine, Serine with 100% of conservation. ď ą Secondary characteristics 1.Luciola mingrelica and Vibrio harveyi have alpha helix, extended strand, beta-turn, and a random coil with little variation in no. of residues associated with it. 2. The residues with a strong propensity to form Strand and Turn structures are found more in Luciferin 4 monooxygenase than Alkanal monooxygenase alpha-beta chain. While Helix forming residues are found more in the Alkanal monooxygenase beta chain than the alpha chain. ď ą Tertiary characteristics 1.Structural Comparison
• The Luciferase protein sequences from Luciola mingrelica and Vibrio harveyi, differ in sequence length by 131 amino acids. Negative residues were found more in both the protein sequence . • The bioluminescence mechanism was carried out due to the interaction between luciferase and luciferin. Serine and Threonine are the residues which play role of active site in Luciola mingrelica and Vibrio harveyi. • According to the literature Survey, the active site in the firefly luciferase was identified from the highly conserved residues. Whereas for Bacterial luciferase the active site needs the presences of all the substrates which was predicted to be present only in the Lux A subunit and function of the beta subunit is to stabilize the active conformation of the structure by specific interactions between conserved residues in the beta subunit and alpha subunit loop region close to the active site
• The interesting phenomenon of light emission was found to be different in Prokaryotic and Eukaryotic organisms. • As per the studies, it was understood that may both the proteins play the same mechanism but they differ in their physical-chemical properties, secondary structure, and residues associated with the active site which affects the intensity of light produced by Bacteria and Firefly. • For Further studies ,as bacteria have a more complex mechanism, so to understand the whole Bacterial bioluminescence mechanism the site of acylation of the Lux gene is needed to be analysed.
•
Structural comparison between lux A and firefly luciferase . They shares 0.56% of identity.
Structural comparison between lux B and firefly luciferase . They shares 1.05% of identity.
2.Identifying Active site of Luciferase protein using Pymol. Determining the physicochemical properties using EXPASY PROTPARAM
• • •
• •
Comparison of the sequence by alignment using BLAST, Clustal omega, Jalview and SOPMA
• • •
Modelling Luciferase 4 monooxygenase using Swissmodel
The residues at positions p.43 Glu, p.75 Ala, p.107 Arg, p.109 Leu, p.175 Glu, p.176 Ser, p.179 Thr on the alpha chain of the Bacterial Luciferase was found to be present at active site for bacterial bioluminescence mechanism.
•
•
Martini, S., Haddock, S. “Quantification of bioluminescence from the surface to the deep sea demonstrates its predominance as an ecological trait.� Sci Rep 7, 45750 (2017). https://doi.org/10.1038/srep45750 Kaskova, Zinaida M., Aleksandra S. Tsarkova, and Ilia V. Yampolsky. “1001 Lights: Luciferins, Luciferases, Their Mechanisms of Action and Applications in Chemical Analysis, Biology, and Medicine.� Chemical Society Reviews 45, no. 21 (2016): 6048–77. https://doi.org/10.1039/C6CS00296J. Lee, John. Bioluminescence, the Nature of the Light, 2015. Suzuki, Kazushi, Taichi Kimura, Hajime Shinoda, Guirong Bai, Matthew Daniels, Yoshiyuki Arai, Masahiro Nakano, and Takeharu Nagai. “Five Colour Variants of Bright Luminescent Protein for Real-Time Multicolour Bioimaging.� Nature Communications 7 (December 14, 2016): 13718. https://doi.org/10.1038/ncomms13718. Brodl, Eveline, Andreas Winkler, and Peter Macheroux. “Molecular Mechanisms of Bacterial Bioluminescence.� Computational and Structural Biotechnology Journal 16 (January 1, 2018): 551–64. https://doi.org/10.1016/j.csbj.2018.11.003. Nackerdien, Zeena E., Alexander Keynan, Bonnie L. Bassler, Joshua Lederberg, and David S. Thaler. “Quorum Sensing Influences Vibrio Harveyi Growth Rates in a Manner Not Fully Accounted For by the Marker Effect of Bioluminescence.� PLOS ONE 3, no. 2 (February 27, 2008): e1671. https://doi.org/10.1371/journal.pone.0001671. Fisher, Andrew J., Frank M. Raushel, Thomas O. Baldwin, and Ivan Rayment. “Three-Dimensional Structure of Bacterial Luciferase from Vibrio Harveyi at 2.4 .ANG. Resolution.� Biochemistry 34, no. 20 (May 23, 1995): 6581–86. https://doi.org/10.1021/bi00020a002. Ugarova NN. “Luciferase of Luciola mingrelica fireflies. Kinetics and regulation mechanism.� J Biolumin Chemilumin. 1989;4(1):406-418. doi:10.1002/bio.1170040155 Hanna, Charles H., Thomas A. Hopkins, and John Buck. “Peroxisomes of the Firefly Lantern.� Journal of Ultrastructure Research 57, no. 2 (November 1, 1976): 150–62. https://doi.org/10.1016/S0022-5320(76)801050. White, E.H., McCapara, F. Field, G.F. & McElroy, W.D.(1961).�The structure and synthesis of firefly luciferin�. Journal of the American Chemical society, 83(10), 2402-2403. Doi:10.1021/ja01471a051 Conti E, Franks NP, Brick P. “Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes�. Structure. 1996;4(3):287-298. doi:10.1016/s0969-2126(96)00033-0
The Eukaryotes possess organs that support the emission of light which is different for different species. Organisms like Luciola mingrelica (Southern Russian firefly) in contrast to the multigene bioluminescence system of bacteria have a simple one enzyme system . Determining the active site of the protein structure using PYMOL
Structural comparison of enzyme forming different Bioluminescent systems using CHIMERA
The residues at positions p.201 Ser, p.247His, p.345 Thr, p.341 GLy, p.318 Gly, p.340 Gln, p.531 Lys, p.424 Asp of firefly luciferase was found to be an active site for Eukaryotic bioluminescence mechanism.
I would like to express my sincere gratitude towards the Post-Graduate Department of Biotechnology , St. Xavier’s College for providing an opportunity to perform an insillico research project. A special mention to my mentor Mrs. Norine D’Souza, and my co-guide Ms. Rutuja Yelmar for their constant guidance and support to carry out this project successfully.