In the previous article, similar gene sequences of an established mercuric ion reductase or merA gene were identified. They were studied from the NCBI database using BLAST tool. In this article, the protein sequence of merA enzyme is studied with respect to its closely related sequences found in NCBI database, through Multiple Sequence Alignment (MSA). MSA refers to the alignment of three or more biological sequences, protein or nucleic acid of similar length.
The purpose of multiple sequence alignment is to characterize protein families. It also determines the consensus sequence of the aligned sequences and reveals biological facts about proteins. For instance, analysis of proteins in the manner of displaying their structures. Thus, MSA is useful in bioremediation studies to understand the protein lineage of an important enzyme or protein. Moreover, the evolutionary relationship of useful proteins and enzymes in bioremediation processes are identifiable across different species. The sequence of interest in this article was derived from E.coli and has been extensively used in mercury phytoremediation studies in the past.
Mode of action of mercury reductase enzyme
Mercury reductase or mercuric ion reductase is an oxido-reductase enzyme and flavoprotein. It catalyses the reduction of the mercuric ion to inert elemental mercury. Furthermore, encoded by merA gene (found in the mer operon), mercury (II) reductase enzyme is present in the cytoplasm of bacteria (Lian et al., 2014). It also reduces Hg2+ ions using Nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate. The figure below shows the mode of action of this enzyme, converting mercuric ion to volatile mercury.
In the catalytic active site of the enzyme, Hg2+ is complex with two cysteine thiolates in a linear geometry. NADPH from the cytoplasm of the cell undergoes a hydride transfer with an embedded Flavin Adenine Dinucleotide (FAD) forming FADH. The resulting FADH– then reduces Hg2+ into Hg0, thereby returning to its original FAD state. Hg0 is then released from the enzyme as a volatile vapour. However, mercury (II) reductase cannot alone reduce organomercury compounds such as methylmercury. In this case, merB cleaves the carbon-mercury bonds of organomercuric compounds via protonolysis and forms a mercury dithiolate complex. MerB then transports Hg2+ directly to merA for reduction (Silva and Rodrigues, 2015).
The aim of the article
This article further explores the merA gene sequence analysed in the previous article. It was adopted by Rugh et al., 1996 for mercury phytoremediation, with respect to its homologous protein sequences. The aim is to determine the functional profile and conserved domains of a selected merA enzyme sequence through multiple sequence alignment. Through this, it is possible to isolate the most conserved regions of the enzyme playing a potential role in its function. This is because the study of conserved regions of enzymes has a major application in genetic engineering. This includes developing mutant enzyme variants. Furthermore, it targets these conserved regions and also uses them in PCR-based screening studies (Thakur, 2003).
For conducting multiple sequence alignment, the researcher analysed the translated merA gene sequence and its 10 homologous protein sequences (from NCBI BLAST). T-Coffee tool identified the conserved regions of this protein sequence. Furthermore, EXPASY Prosite recognised the important domains.
Multiple sequence alignment
As shown in the figure above, VIRT12233 represents the translated merA gene sequence (protein of interest). The other 10 sequences are homologous sequences originating at NCBI BLAST. Moreover, sequences run in T-Coffee for MSA revealed an overall 99 score for all 11 sequences. This suggests a good alignment. This also suggests that the homologous MerA sequences belonging to different bacterial species were highly conserved. Further evaluation of aligned sequences show approximately 50-aa length sequence being highly conserved. It was signified by the presence of ‘*’ in the output.
Of these 11 sequences, the protein of interest (VIRT12233 in the figure above), is significantly diverse from the other 10 homologous sequences. This is visible in the alignment patterns. However, among the 10 homologous sequences, the MerA sequence belonging to E.coli (ASL56501) was more diverse than the other 9 sequences. Therefore, this sequence does not require further analysis.
Domain recognition in sequences
The researcher submitted a total of 10 sequences, including the protein of interest to EXPASY Prosite for domain recognition based on profiles (domains) and patterns (motifs). As seen from the figure below, 9 sequences identified 25 hits (both motifs and domains). However, EXPASY could not detect any domain or motif in the primary protein of interest. This could be due to its short sequence length. Of the 25 domains it recognised, two main types of hits are evident; heavy-metal associated domain profile (HMA_2) and Pyridine nucleotide-disulphide oxidoreductases class-I active site (PYRIDINE_REDOX_1).
HMA_2 is a domain of 70 amino acids responsible for transporting or detoxifying heavy metals using 2 cysteine residues. PYRIDINE_REDOX_1 active sites contain a pair of cysteine residues. They are responsible for transferring and reducing equivalents from FAD co-factors to the substrates. Also, they are highly conserved around the cysteine residues. These two domains are therefore important in contributing towards the mode of action of the MerA enzyme. However, since no such domains exist in the protein of interest, further analysis will confirm the functional profile of the same.
Importance of merA genes
The protein of interest, used by Rugh et al., (1996) for mercury phytoremediation was studied here. The aim was to determine its conserved domains and its functional profile by means of multiple sequence alignment and domain recognition. Two primary domains, heavy metal associated domain and Pyridine nucleotide-disulphide oxidoreductases class-I active site was found among all the homologous merA protein sequence. Therefore, these domains are important in merA enzyme’s functioning. Future studies on the selection of merA enzyme candidates require study of these domains for appropriate selection of a protein variant. The next article deals with the study of genes and proteins with respect to their functions through the concept called gene ontology. It is a method of standardised description of genes and gene products in all the databases that can reduce error and improve the transfer of knowledge among similar genes and proteins.
- Lian, P. et al. (2014) ‘X ‑ ray Structure of a Hg 2+ Complex of Mercuric Reductase (MerA) and Quantum Mechanical/Molecular Mechanical Study of Hg 2+ Transfer between the C ‑ Terminal and Buried Catalytic Site Cysteine Pairs’, Biochemistry, 53, pp. 7211–7222.
- Rugh, C. L. et al. (1996) ‘Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene’, Proceedings of the National Academy of Sciences, 93(8), pp. 3182–3187.
- Silva, P. J. and Rodrigues, V. (2015) ‘Mechanistic pathways of mercury removal from the organomercurial lyase active site’, PeerJ, 3, p. e1127. doi: 10.7717/peerj.1127.
- Thakur, I. S. (2003) Environmental Biotechnology: Theory and Application. New Delhi, India: I K International Pvt. Ltd.
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