1. INTRODUCTION:

Researchers have found a wealth of new information about organisms known as "extremophiles," which are those that are able to survive in environments of extreme cold and heat alkalinity or salinity. Since their discovery, these species have been given a stimulating and challenging environment for study ^1,2.

Exotic habitats that were more common on the primordial Earth are where these severe microbial growth conditions can be found today. When organic solvents or heavy metals are found to be present is also common in extreme settings. Increasing numbers of studies are being conducted on the evolution and taxonomy of extreme-environment organisms, such as thermophiles and Pscryophiles ³.

Warm water seeps from the Earth in the form of a spring. However, no universally agreed-upon definition exists for what constitutes a "hot spring." ⁴ described hot springs merely as warm groundwater that flows naturally to the ground. described hot springs as springs where the water temperature is substantially above that region's average annual air temperature ⁵.

As a potential source of thermophilic bacteria, several hot springs in the area were investigated. As a result of the wide range of hot springs and the fact that most microbiologists attempt to extract thermophiles from hot environments, beneficial enzyme-generating bacteria have been found. Biochemical and molecular techniques can be used for both the bacteria and their enzymes ⁶. Consequently, there is a growing need for enzymes capable of operating in a variety of industrial conditions and that can handle various production processes. Throughout the ages, as DNA and protein technology has advanced, many ways have been devised to meet these ever-increasing demands. Bioprospecting is one of these methods, which entails searching for new enzymes in various environments with a high amount of natural variation in microorganisms⁷. For discovering these enzymes, metagenomics is also an essential new technique, and this mining strategy for the biotechnology and pharmaceutical industries has been revealed as a promising one ⁸. Metagenomics has two words; Meta(analysis), a statistical analysis method of the outcomes of two different analyses, and Genomics, which is the genetic makeup analysis ⁹. Genomic DNA is extracted from environmental samples and sequenced using high-throughput methods such as shotgun sequencing and 454 pyrosequencing, which reduces the failure of essential organisms during cultivation. Analysis of gene or species richness and distribution is generally done using sequence-based screening, whereas functional-based screening is used to evaluate the functional capabilities of microbial communities ¹⁰.

The protease enzyme is one of the essential enzymes in the body. Proteases are degradative enzymes that hydrolyze the peptide bond found in the amino acid sequence of polypeptides, allowing the polypeptide to be broken down. Peptides from microorganisms are the most often utilized enzymes in a wide range of industrial applications such as detergents and animal feed and leather, textiles, trash, and therapeutic applications ¹¹. Depending on their pH, they are classified as either acidic, neutral, or alkaline. Proteases are classified as endopeptidases or exopeptidases based on the location of their active site on the protein substrate. Additionally, they can be classed as metal, cysteine, aspartic, or serine proteases based on their catalytic properties ^{12, 13}. The rhomboid type was taken into consideration in our research. Even though it is a large family of associated membrane proteins with a diverse range of biological functions, they all share the same catalytic core domain, composed of six membrane-spanning segments containing several highly conserved sequence motifs that are characteristic of the family. Rhomboid proteases have their active sites incorporated in the lipid bilayer where their substrate is hydrolyzed to or adjacent to the transmembrane domains (TMDs) ^14,15.

The primary purpose of this study is to conduct a thorough examination of the genes linked to Rho activity in bacteria found in hot springs. The ultimate goal is to gain a deeper understanding of the genetic mechanisms that underlie this enzymatic activity. This involves the discovery of distinct genes that are accountable for the function of rhomboid proteases, followed by a comparative analysis of these genes. This study entails the examination of gene sequences in order to determine if differences in enzyme temperature ranges are a result of changes in the general structure of polypeptides or adjustments to catalytic sites. Additionally, it aims to comprehend the importance of amino acid composition in maintaining protein stability. Moreover, the present study aims to assess protein domains across various sequences in order to ascertain putative functional domains. In addition, this study aims to employ computational techniques in order to forecast the three-dimensional configuration of rhomboid proteases and evaluate their secondary structure, solvent accessibility, and B factor. The ultimate goal entails performing docking investigations to predict the protolytic and aminopeptidase functions of certain rhomboid proteases, with the intention of aiding future endeavors in protein engineering to improve psychrophilic and thermophilic properties. This work seeks to provide useful insights into the molecular processes that underlie the function, evolution, and possible therapeutic applications of rhomboid proteases through a series of comprehensive investigations.

2. MATERIALS AND METHODS:

2.1 Data collection for Rho from JGI and PATRIC site:

Rho gene of bacteria isolated from hot springs was obtained from the Joint Genome Institute (JGI) data base ¹⁶. We considered the gene ID, isolate name, DNA sequence length, and amino acid length. After eliminating the redundancy, non-redundant sequences were obtained using Cluster Database at High Identity with Tolerance (CD-Hit) (http://weizhong-lab.ucsd.edu/cdhit-web-server/cgi-bin/index.cgi?cmd=Server%20home). Ninety percent sequence identity cut-off was considered for clustering the sequences ¹⁷.

2.2 Gene sequence alignment of Rho using MAFFT and MEGA X:

Rho gene sequences of bacteria isolated from hot springs were aligned with the MAFFT website http://mafft.cbrc.jp/alignment/server/ and phylogenetic tree was constructed by the use of Mega X v 11.0 sequence analysis software and Tree Of Life (http://itol.embl.de) is a web-based tool¹⁸ ¹⁹ ²⁰.

2.3 Prediction of Rho amino acid content using expasy:

The ExPASy server has a utility called ProtParam that may be accessed online. It may analyze a protein sequence obtained from Swiss-Prot or TrEMBL or a user-entered. 27 Rho gene sequences were isolated from hot springs to determine the amino acid structure and composition. FASTA sequences of the selected Rho gene were submitted to a web server for analysis, and the results were viewed in this study ²¹.

2.4 Prediction of Rho active domain using interproscan:

The InterProScan (http://www.ebi.ac.uk/InterProScan) was used to evaluate the protein domains of all sequences 27 Rho gene sequences were isolated from hot springs were submitted to analyze the conserved motifs of the encoded proteins ²².

2.5 Prediction of 3d structure for Rho using I-TASSER:

The Rho gene sequence of the hot spring isolated strain was submitted to homology modeling by the I-TASSER Server for 3D-structure prediction. After the structure assembly simulation, I-TASSER uses the TM-align structural alignment program to match the first I-TASSER model to all structures in the PDB library²³.

2.6 Improvement of 3d structure Rho using modrefiner:

ModRefiner (https://zhanglab.ccmb.med.umich.edu/ModRefiner/) was utilized. In my study, the Rho gene sequences extracted from hot springs was submitted to the ModRefiner server. This process was repeated five times until a tridimensional model corroborated the consensus secondary structure and domain boundary predictions possessed, RMSD and TM-score within range ^24,25.

2.7 Prediction binding site for Rho using dogsitescorer:

The modeled Rho gene from hot-springs isolates were submitted to DoGSiteScorer (https://bio.tools/dogsitescorer):server to prediction active site and obtained the possible binding sites ²⁶.

2.8 Ligand design using chemdsketch and chem3d ultra:

2D structures of the Methionine, Leucine, Rivastigmine, N-carbobenzyloxy-L-alanine and 4- amidino phenyl methanesulfonyl fluoride compounds were generated with chemdsketch v 11.0, a software ²⁷. These ligands were then copied into Chem3D ultra v 16.0, which was used to generate a 3D model. Molecular mechanics was then used to minimize the amount of energy required to run the model (MM2) . These structures with the lowest possible energy expenditure are considered for docking ^{28, 29}.

2.9 Energy minimization of Rho using chiron server:

The Rho sequences for hot springs have been uploaded to Chiron (https://dokhlab.med.psu.edu/chiron/processManager.php) The clashes to resolve were selected from relevant work areas, and results were delivered within 24 hours ^{30, 31}.

2.10 Substrates for molecular docking studies using CB-DOCK:

The 3D structures of substrates like methionine, leucine, rivastigmine, N-carbobenzyloxy-L-alanine and 4-amidino phenyl methanesulfonyl flurode were docked with the binding sites of modeled Rho from the isolates by using CB-Dock Server (http://clab.labshare.cn/cb-dock/php/blinddock.php) ^{32, 33}.

2.11 Prediction of ligand-Rho interactions using discovery studio:

The interactions between methionine, leucine, rivastigmine, N-carbobenzyloxy-L-alanine and 4- amidino phenyl methanesulfonyl fluoride and Rho from the isolates as docked complex were analyzed by Discovery Studio (http://www.adrianomartinelli.it/Fondamenti/Tutorial_0.pdf) ³⁴.

3. RESULT:

Hot springs isolates collections and analysis of rhomboid gene:

JGI offers comprehensive high-throughput sequencing, design, and synthesis of DNA, metabolomics, and computational methods that allow system based scientific approaches to these challenges. While (IMG) introduces a way for comparative analysis of predominantly microbial genomes, the program also supports samples from the environment. The purpose is to promote genome visualization and exploration from a practical and evolutionary point of view ¹⁶. CD-HIT is a widely used technique for minimizing sequence duplication and is regarded as the most advanced method available³⁵. From the JGI site, 93 Rho gene sequences from bacteria retrieved from hot springs samples, after removing redundancies by the CD Hit program, and according to the number of amino acids, 27 Rho gene sequences were chosen for the study (Please see figure 1). all the genomic sequences of bacteria, archaea, and eukaryotes, except some small genome’s organisms. Based on laboratory experiments done on Drosophila and AarA protein of the bacterium Providencia stuartii to study the developmental regulator rhomboid, rhomboids are believed to be intramembrane serine proteases, the signaling mechanism of which is retained in eukaryotes and prokaryotes ³⁶. According of (Figure 2 (a, b)) shows amino acid sequences of the Rho highly conserved, the highlighted boxes also indicate some samples of conserved areas in amino acid sequences. Phylogenetic tree Using Neighbor-Joining, the evolutionary history of hot springs isolates was inferred. The ideal tree is illustrated. Next to each branch is the percentage of 500 replicate trees in which the relevant taxa clustered together in the bootstrap test. (Alongside the branches) The evolutionary distances were calculated using the Poisson correction method and are expressed in terms of the number of substitutions of amino acids per site. Twenty-six Rho sequences were analyzed based on the hot spring isolates. The final dataset had 562 locations.

Comparison of amino acid frequencies in Rho isolated from extremophile bacteria:

In general, thermophilic enzymes alter their amino acid composition to withstand high temperature, and this includes additional charged residues providing more ionic interactions that will stabilize the structure, more aromatic residues that stabilize the protein core, fewer uncharged polar residues, fewer amides amino acids, and more hydrogen bonding and salt bridges. In addition, increase, hydrophobic residues in the protein core maintain tight molecular packing, higher hydrophobic core, lower backbone, and entropy of the side chains ³⁷, ³⁸. In comparison, cold-adapted proteins have a variety of modifications in amino acids that offer increased flexibility, including lower salt bridges and hydrogen bonds, lower proline residue content, lower Arg/(Arg + Lys) ratio, higher serine and glycine availability, more asparagines, increase in the negative net charge, broad accessible hydrophobic surfaces, and longer loops ³⁹,⁴⁰. In our study, the aliphatic amino acids are predominant (the amino acids that contain alkyl (CH2) group) in rhomboid protease; Leu come in the first place, and Ala in the third place, followed by Ile and Val, these hydrophobic non-polar amino acids play an essential role in the hydrophobic activity, which is the critical factor in maintaining conformational stability within the protein's inner and outer portions (Please see figure 3). Gly is the second most presented amino acid. It is a hydrophobic amino acid with no side chain, or we can consider the hydrogen ion as its side chain. This gives the Gly more conformational flexibility. Ser is a polar amino acid with a hydroxymethyl group at its active site, which makes it capable of forming hydrogen bonds with a number of polar substrates; it is presented in protein active sites as the classical Asp-His-Ser catalytic triad responsible for the hydrolysis of other molecules ⁴¹. Histidine is a polar positively charged amino acid with imidazole side chain, it’s percent 2.09 in hot springs isolates, its presence is widely common in metal-binding sites (like zinc) of the proteins; also it is presented in the catalytic triads in which its basic nitrogen abstract a proton from serine, threonine, or cysteine to activate it as a nucleophile ⁴².

Figure 3. The orange column displays the average amino acid ratio in a Rho Hot-springs isolates.

Identification of active domain for rhomboid protease:

The rhomboid family, S54, has been little explored. Researchers have discovered this phenomenon in bacteria, archaea, and more recently, eukaryotes, beginning with Drosophila melangolaster ⁴³. InterProScan results in (figure 4) have shown various domains of the proteins determined from the isolates; Peptidase S54-rhomboid domain is the predominant domain in Rho isolated from hot springs. The rhomboid family (S54) is a family that has received limited research attention. The presence of this phenomenon has been extensively seen in bacteria, archaea, and more recently, eukaryotic creatures, with the inaugural discovery in Drosophila melangolaster ⁴⁴.

Molecular docking:

Molecular docking is a computational technique in drug design used to predict the binding mode of small drug-like molecules within the active site of an enzyme or cell surface receptor binding pocket. This can be done by placing the molecules in a "docking station," which simulates the enzyme's active site. Through the use of molecular modeling, we can view the protein active site with ligands in a variety of different orientations and conformations. In addition, it enables us to rank the various ligands based on their binding affinity, which is graded according to the ideal binding geometries and energies of the individual molecules. This ranking can then determine which ligands are the most effective. ^{67, 68} In the current investigation, all docking tests were carried out using the CB-Dock server, discover studio. The docked conformation of 4- Amidino Phenyl Methanesulfonyl Fluoride, Leucine, Methionine, N-Carbobenzyloxy-L-Alanine and Rivastigmine (ligands) with the active conformation of Rho (receptor) isolated hot-springs bacteria (please see figure 11a, 11b, 11c, 11d and 11e) revealed that numerous potential interactions were present. The vina score of ligands compared after interaction with studied Rho for Hot Spring bacteria were present in (Table 1).

Evaluation of extremophiles Rho- ligand interaction:

2D interaction of 4- Amidino Phenyl Methanesulfonyl Fluoride with Rho was isolated from hot-spring bacteria (please see figure 11.a). It showed (Gly) 17, TYR 31, and ILE number 220 forming conventional hydrogen bonds represented in dark green dotted lines. In addition, TYR 31, ILE number 220 formed Pi-Pi stacked and Pi-sigma bonds respectively represented in dark pink color and purple color, VAL 148 and PRO number 43 formed Pi-Alkyl bond, GLY 42 formed Pi-sulfur bond represented in dark yellow color. Also, van der Waals non-bonded interactions in light green with a vina score (-7.2). The 2D interaction of leucine with Rho was isolated from hot springs (see figure 11b). ARG 212, LYS 274, MET 157, LEU 256 forming conventional hydrogen bonds represented in dark green dotted lines. On the other hand, GLU 2 forms Attractive charge bonds represented in orange dotted lines. Also, LEU 256, 278, and ARG 260 Alkyl bonds are represented in pink dotted lines. Van der Waals non-bonded interactions in light green with a vina score (-5.3). It shows interaction of methionine interaction with Rho was isolated from hot springs (see figure 11.c). It shows the ARG 260 forming conventional hydrogen bonds represented in dark green dotted lines. On the other hand, ARG 260 and LEU (278, 259) form alkyl bonds represented in pink dotted lines. Also, van der Waals's non-bonded interactions are in light green with a vina score (-4.1). The interaction of N-Carbobenzyloxy-L-Alanine with Rho was isolated from hot springs (see figure 11d). It shows SER 50 forming conventional hydrogen bonds represented in dark green dotted lines. In addition, ILE number 220 forms Pi-sigma bonds represented in purple color, TYR 31 forming Pi-Pi stacked represented dark pink color, VAL 148 and LEU 223 forming Pi-Alkyl bond represented pink color, GLY 42 forming Pi-sulfur bond represented in dark yellow color. Also, van der Waals non-bonded interactions in light green with a vina score (-7.8). The interaction of Rivastigmine with Rho isolated from hot springs (see figure 11e) revealed five Carbon Hydrogen bonds depicted by light green dotted lines: VAL 148, GLN (219, 216), TYR 31, and GLN 151. In addition, ILE number 220 forms Pi-sigma bonds depicted in purple, TYR 31 forms Pi-Pi stacking depicted in dark pink, VAL 51, 148, and PRO 43 form Alkyl and Pi-Alkyl links, respectively, depicted in pink, and GLY 42 creates Pi-sulfur bond depicted in dark yellow. In addition, van der Waals non-bonded interactions are represented in light green with a vina score (-6.9). The aminopeptidase activity was determined by docking the amino acids like Leucine and Methionine. Unfavorable bonds affect the activity stability of the ligand. The formation of any unfavorable bond between/in the protein-ligand complex reduces the stability of the complex as these types of bonds indicate a force of repulsion between 2 molecules and an atom. ⁶⁹ The Leucine interaction with Rho isolated from a Hot spring form a stable complex structure by forming four conventional hydrogen bonds. Furthermore, the Rho inhibitors such as Rivastigmine, N-carbobenzyloxy-L-alanine, and 4-amidino phenyl methane sulfonyl fluoride ⁷⁰ were docked by CB-Dock to understand the proteolytic activity of the predicted Rho model from Hot spring bacteria. The docking interactions between the binding site residues and the compounds with their respective docking (vina) scores were given, also visualized in discovery studio, to compare and validate the docking results. The docking output was the free binding energy with which the ligand binds to the pocket of the receptor protein. The highest scoring ligand (4- amidino phenyl methane sulfonyl fluoride, N-Carbobenzyloxy-L-Alanine, and Rivastigmine) gave a better binding affinity score of -7.2, -7.8, and -6.9, respectively, moreover, three ligands demonstrated pi-sulfur bond with GLY 42 and Pi-stacked with TYR 31 for Rho isolated from Hot spring isolate (see Table 1). The docking studies showed that critical amino acid residues in Rho isolated from Hot spring bacteria were VAL (148), ILE (220), GLY (42), and TYR (31). Rho isolated from Hot spring bacteria demonstrated conventional hydrogen bond, Carbon Hydrogen bond, Pi-sulfur with inhibitor ligands. Probably these structures are the most stable interaction energy.

CONCLUSION:

In conclusion, the rhomboid gene sequence showed a lot of similarities between the hot spring isolates. It is not clear how this protein has changed at high and low temperatures, but the fact that this family is found in almost all bacteria, archaea, and eukaryotes suggests that this protein is part of the last common ancestor. Phylogenetic tree analysis studies indicate a likely bacterial or archaeal origin. Different amino acids have different functions, and the presence of active domains determines the protein's function. By looking at the isolates' amino acids and active domains, we found that their structure and function are very similar to those of other living things. In fact, the main structure and functional sites of rhomboids stay the same in almost all living things. Good selection may have retained the protein's proteolytic activity. Rhomboid proteases are released by many bacteria, but their biological activity is unknown due to the lack of endogenous substrates. In vitro, various bacterial rhomboids have been shown to process eukaryotic rhomboids using a typical enzyme mechanism. Rhomboid intramembrane proteases are gaining popularity in infectious diseases and cancer. Rhomboid intramembrane proteases rapidly link to Parkinson's disease, cancer, type II diabetes, and bacterial and malaria infections. Recent discoveries about rhomboids' structure and enzyme activity, as well as efforts to identify inhibitors, are shedding light on their potential as medicinal targets.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

I like to extend my sincere appreciation to the esteemed faculty members and academic advisors who have offered invaluable assistance, constructive critique, and unwavering support throughout the entirety of my academic endeavors. The individual's proficiency, sagacity, and commitment to instruction and guidance have played a pivotal role in molding my aptitude for research and academic endeavors.

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Received on 24.04.2024 Revised on 30.07.2024

Accepted on 06.10.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5890-5900.

DOI: 10.52711/0974-360X.2024.00894

ligands Name	Vina score	Cavity size	center			size
ligands Name	Vina score	Cavity size	X	Y	Z	X	Y	Z
4- amidino phenyl methanesulfonyl fluoride	-7.2	88	50	55	65	19	19	19
leucine	-5.3	81	56	66	78	17	17	17
methionine	-4.1	81	56	66	78	17	17	17
N-Carbobenzyloxy-L-Alanine	-7.8	282	59	52	49	21	21	21
rivastigmine	-6.9	282	59	52	49	20	20	20