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Antifreeze proteins (AFPs) are the class of polypeptides, facilitating cold-adaptation in psychrophilic organisms, whereas, Choline dehydrogenase (CHDH), and Betaine-aldehyde dehydrogenase (BADH) catalyze the production of a common osmoprotectant, glycine betaine, which owes for organism’s freeze tolerance as well. The present Bioinformatics study selects three proteins: type 1 AFP, CHDH, and BADH from an extreme psychrophile, Psychromonas ingrahamii 37 (Pi37) and performs a systematic in-silico analysis to shed light on the evolutionary as well as the functional aspects of these proteins. Multiple sequence alignment (MSA) shows type 1 PiAFP with very low, whereas, PiCHDH and PiBADH with high residue conservation across BLASTp homologs, and phylogenetic trees generated through CLC workbench presented evolutionary perspective on query proteins. MSA identifies key regulatory residues/patterns in query proteins that are evolutionarily conserved across BLASTp organisms, which suggest their functional significance. Primary and secondary structure analysis has been performed. Conserved domain analysis predicts that type 1 PiAFP, PiCHDH, and PiBADH belong to Band_7_5, GMC _C-terminal, and ALDH_F9_TMBADH protein families, respectively, and their possible functions have been inferred. GMC oxidoreductase signature 1 and 2, and ALDH cysteine and glutamic acid are catalytic sites in PiCHDH and PiBADH, respectively. Homology modeling of these protein and their domains provide a fairly good hypothetical models of the query proteins and their functional domains for the future investigations. Protein-protein interaction prediction, using STRING, yields functional partners of query proteins. The present work is an initial step towards comprehending the molecular basis of extreme adaptation of Pi37,and additional works are required, in light of the significant data and inferences presented here, for complete understanding of the structure and function of these proteins, which may have bearing on the extreme psychrophily and halophily of Pi37.
Antifreeze proteins; computational analysis; homology modeling; osmoregulation; protein domains; protein-protein interaction, proteomic tools.
Psychrophiles have been studied primarily to understand biological mechanisms of adaptation to extremely cold conditions. In reaching for an understanding of how life processes work at extremes of temperature, most of the focus to date has been on the properties of enzymes of extremophiles1, 2.Recently, few studies on the sea ice bacterium Pi37 have illustrated the species characterization3, whole genomics4, subfreezing growth6, and enzyme catalyzed cold-adaptation7 of this extreme psychrophile. The extreme psychrophile, Pi was isolated from sea ice from the Arctic4. Strain 37T was considered to represent a novel species, which was provisionally named Pi sp.nov1. It grows exponentially with a doubling time of 240 hours at -12°C and may well grow at even lower temperatures6. It has been found that cold-hardy organisms possess some proteins and enzymes which are active at low temperatures7, and facilitate their survival in such extreme environments.
AFPs were initially characterized in marine fishes8,9 where they protect their hosts from freezing by binding to, and preventing the growth of, sea ice crystals10. AFPs lower the freezing temperature of a solution containing ice below the melting point of the ice. This difference between the freezing and melting temperatures is called thermal hysteresis (TH) and is used as a measure of antifreeze activity. AFPs were subsequently found in freeze-tolerant organism11,12 where, rather than preventing freezing, they stop ice crystals in frozen tissues from growing larger through the process of ice recrystallization (IRI)13. Another feature of a typical psychrophile is its ability to produce an intracellular solute to counterbalance the osmotic effect of higher salinities in brine pockets where organisms reside that are encountered as sea ice freezes 14. Bacteria and plants often accumulate choline, since it can easily be converted to glycine betaine, a common osmoprotectant 15. While the conversion of choline to glycine betaine for the purpose of accumulating the latter as an osmoprotectant is considered biosynthetic, the conversion of choline to glycine betaine in this pathway is performed by the action of two dehydrogenases –CHDH and BADH. When the bacteria grow at normal osmolarity conditions, they degrade choline, using it as a carbon and nitrogen source. However, under inhibiting osmotical conditions enzyme activities, which lead to glycine betaine degradation, decrease, while enzyme activities that convert choline to glycine betaine either remain constant or increase. In this way, a high concentration of glycine betaine can be maintained in osmotically stressed cells16. Hence, the present computational analysis and homology modeling of type 1 PiAFP, PiCHDH, and PiBADH may provide essential insights into the molecular basis of cold-adaptation and osmoprotection in Pi37, and the information reported here may open avenues to further explorations of the grounds of unique adaptation of this bacterium.
The study of unique survival mechanisms of extremophilic microorganisms are of great interest to microbiological community. Pi37 is an extreme psychrophile and not much has been understood about its adaptive strategies yet. The present work endeavors to provide evolutionary and functional insights through in-silico study of three proteins: type 1 PiAFP, PiCHDH, and PiBADH from Pi37, which may have bearing on the psychrophily and halophily of this bacterium. Phylogenetic analysis infers that evolutionary proximity between type 1 PiAFP and type 1 AFP of Paenibacillus lactis 154, with very low sequence identity, may be an outcome of functional convergence; on the other hand, high degree of sequence conservation and close phylogenetic relationship between PiCHDH and CHDH of Moritella sp. PE36, and between PiBADH and BADH of Shewanella frigidimarina, BADH of Bermanella marisrubri, and BADH of Colwellia psychrerythraea 34H suggest divergent evolution of these proteins from a common ancestor. MSA identifies some conserved patterns/residues in each query protein that are evolutionary preserved across BLASTp species and imply their functional significance. Physicochemical characterization studies provided essential data about the properties of target proteins, such as pI, EC, AI, GRAVY and Instability Index; it revealed that all query proteins are glycine-rich and hydrophilic in nature. Type 1 PiAFP, and PiCHDH have more coiling, whereas, PiBADH is α-helical; as well as type 1 PiAFP and PiCHDH have been found to have 3 most probable SS bonds, while no SS bond is predicted in PiBADH. Present study found three fairly good representative homology models of type 1 PiAFP (3bk6), PiCHDH (3t37) and PiBADH (1a4s), which was again validated by homology modeling of domains through Phyre2. Although energy-minimization has not been performed here, the model information is significant and can further be utilized to conduct in-depth evolutionary, structural, and functional analysis of these proteins from Pi37.
Conserved domain analysis is integral to unearth functional aspects of proteins by knowing their family history, thus, significant inferences about possible roles ofquery proteins in environmental adaptations of Pi37 have been drown here, which need to be further analyzed in future studies. Some putative sites of posttranslational-modifications have been configured in all query proteins that may provide functional insights; and catalytic sites, such as GMC_OxRdtase signature 1and 2, and Aldedh cystein and glutamic acid in PiCHDH and PiBADH, respectively, have been confirmed and located in domain models. Further, the paper uses a standard resource (STRING) to predict different proteins that bind to the proteins of interest and utilizes the information to assume the possible functions of target proteins.
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