Probiotics, prebiotics and synbiotics: Safe options for next-generation therapeutics.

Probiotics have been considered as an economical and safe alternative for the treatment of a large number of chronic diseases and improvement of human health. They are known to modulate the host immunity and protect from several infectious and non-infectious diseases. The colonization, killing of pathogens and induction of host cells are few of the important probiotic attributes which affect several functions of the host. In addition, prebiotics and non-digestible food substances selectively promote the growth of probiotics and human health through nutrient enrichment, and modulation of gut microbiota and immune system. This review highlights the role of probiotics and prebiotics alone and in combination (synbiotics) in the modulation of immune system, treatment of infections, management of inflammatory bowel disease and cancer therapy. KEY POINTS: • Probiotics and their derivatives against several human diseases. • Prebiotics feed probiotics and induce several functions in the host. • Discovery of novel and biosafe products needs attention for human health.

A Study on Knowledge and Awareness of Cervical Cancer Among Females of Rural and Urban Areas of Haryana, North India.

Lack of awareness of screening methods, risk factors, and symptoms may lead to late diagnosis and poor prognosis of cervical cancer. The plan of this study was to assess the level of awareness about cervical cancer and HPV vaccine among females of rural and urban areas of Haryana, India. This cross-sectional study was performed using a comprehensive self-designed questionnaire on 1500 women of urban (700) and rural (800) background aged 18-65 years, evaluating their knowledge for cervical cancer and screening, HPV infection and its preventive measure, and symptoms and risk factors. Data obtained was analyzed and interpreted by using simple percentages and bar charts. Most of the participants were aged between 21 and 30 years and had college level education. Majority of the women from rural areas had poor knowledge about cervical cancer (55%) and its screening (75%), HPV infection (87.5%), and HPV vaccine (95%) compared with urban areas. Knowledge about symptoms and risk factors was very low in both rural and urban areas. Whatever little knowledge the women had about cervical cancer was from college education, friends, neighbors, relatives, and medical practitioner or doctors. The survey pointed to the critical need to educate women about cervical cancer and its early diagnosis, related risk factors, symptoms, and preventive measures which can be achieved by launching extensive awareness programs for educating females about cervical cancer in India.

Catalytic diversity and homotropic allostery of two Cytochrome P450 monooxygenase like proteins from Trichoderma brevicompactum.

Trichothecenes are the secondary metabolites produced by Trichoderma spp. Some of these molecules have been reported for their ability to stimulate plant growth by suppressing plant diseases and hence enabling Trichoderma spp. to be efficiently used as biocontrol agents in modern agriculture. Many of the proteins involved in the trichothecenes biosynthetic pathway in Trichoderma spp. are encoded by the genes present in the tri cluster. Tri4 protein catalyzes three consecutive oxygenation reaction steps during biosynthesis of isotrichodiol in the trichothecenes biosynthetic pathway, while tri11 protein catalyzes the C4 hydroxylation of 12, 13-epoxytrichothec-9-ene to produce trichodermol. In the present study, we have homology modelled the three-dimensional structures of tri4 and tri11 proteins. Furthermore, molecular dynamics simulations were carried out to elucidate the mechanism of their action. Both tri4 and tri11 encode for cytochrome P450 monooxygenase like proteins. These data also revealed effector-induced allosteric changes on substrate binding at an alternative binding site and showed potential homotropic negative cooperativity. These analyses also showed that their catalytic mechanism relies on protein-ligand and protein-heme interactions controlled by hydrophobic and hydrogen-bonding interactions which orient the complex in optimal conformation within the active sites.

Spin Inversion Phenomenon and Two-State Reactivity Mechanism for Direct Benzene Hydroxylation by V4O10 Cluster.

The direct and selective introduction of hydroxyl group into aromatic compounds remains one of the challenging problems in oxidation chemistry. Keeping in view the reported reactivity of vanadium oxide in C-H activation of saturated hydrocarbons, the study explores the reactivity of neutral V4O10 cluster with benzene through rigorous computations performed within the formalism of density functional theory. Three possible reaction channels for the reactivity of V4O10 cluster with benzene have been deciphered, and comprehensive understanding of all possible mechanistic pathways has been obtained by analysis of all the intermediates and transition states encountered en route. The study provides promising evidence of direct abstraction of hydrogen by terminal oxygen of the cluster via three-centered transition state. The scan of potential energy surfaces for the reactivity of the cluster in its ground (singlet) and first excited (triplet) spin multiplicity states establishes two-state reactivity mechanisms. The spin crossover point has been identified through geometric and thermodynamic parameters, partial charges, and intrinsic reaction coordinate calculations. The study establishes the efficacy of V4O10 cluster species in direct hydroxylation of benzene to phenol.

Multifaceted impact of trichothecene metabolites on plant-microbe interactions and human health.

Fungi present in rhizosphere produce trichothecene metabolites which are small in size and amphipathic in nature and some of them may cross cell membranes passively. Hypocreaceae family of rhizosphere fungi produce trichothecene molecules, however it is not a mandatory characteristic of all genera. Some of these molecules are also reported as growth adjuvant, while others are reported as deleterious for the plant growth. In this review, we are exploring the roles of these compounds during plant-microbe interactions. The three-way interaction among the plants, symbiotic microbial agents (fungi and bacteria), and the pathogenic microbes (bacteria, fungi) or multicellular pathogens like nematodes involving these compounds may only help us to understand better the complex processes happening in the microcosm of rhizosphere. These metabolites may further modulate the activity of different proteins involved in the cell signalling events of defence-related response in plants. That may induce the defence system against pathogens and growth promoting gene expression in plants, while in animal cells, these molecules have reported biochemical and pharmacological effects such as inducing oxidative stress, cell-cycle arrest and apoptosis, and may be involved in maintenance of membrane integrity. The biochemistry, chemical structures and specific functional group-mediated activity of these compounds have not been studied in details yet. Few of these molecules are also recently reported as novel anti-cancer agent against human chondrosarcoma cells.

Leishmaniasis: Omics Approaches to Understand its Biology from Molecule to Cell Level.

Leishmaniasis is the second deadliest vector-borne, neglected tropical zoonotic disease and is found in a variety of clinical forms based on genetic background. Its endemic type is present in tropical, sub-tropical and Mediterranean areas around the world which accounts for a lot of deaths every year. Currently, a variety of techniques are available for detection of leishmaniasis each technique having it's own pros and cons. The advancing next-generation sequencing (NGS) techniques are employed to find out novel diagnostic markers based on single nucleotide variants. A total of 274 NGS studies are available in European Nucleotide Archive (ENA) portal (https://www.ebi.ac.uk/ena/browser/home) that focused on wild-type and mutated Leishmania, differential gene expression, miRNA expression, and detection of aneuploidy mosaicism by omics approaches. These studies have provided insights into the population structure, virulence, and extensive structural variation, including known and suspected drug resistance loci, mosaic aneuploidy and hybrid formation under stressed conditions and inside the midgut of the sandfly. The complex interactions occurring within the parasite-host-vector triangle can be better understood by omics approaches. Further, advanced CRISPR technology allows researchers to delete and modify each gene individually to know the importance of genes in the virulence and survival of the disease-causing protozoa. In vitro generation of Leishmania hybrids are helping to understand the mechanism of disease progression in its different stages of infection. This review will give a comprehensive picture of the available omics data of various Leishmania spp. which helped to reveal the effect of climate change on the spread of its vector, the pathogen survival strategies, emerging antimicrobial resistance and its clinical importance.

Repurposing small molecules of Tephrosia purpurea against SARS-CoV-2 main protease.

Coronavirus infection is a communicable disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged as a global pandemic with deteriorating effect on the world's population. Main protease (Mpro) of SARS-CoV-2 plays a significant role in the viral replication, transcription and disease propagation as well as a potential candidate for drug discovery and development for COVID-19 infection. The current study employed state of art structure-based drug discovery to decipher the role of phytochemicals of Tephrosia purpurea against Mpro. Tephrosia purpurea is being used as a traditional medicinal plant for the treatment of cough, breathlessness and fever as per the Indian Materia Medica. Screening of the phytochemicals of Tephrosia purpurea against Mpro was performed using molecular docking approach to identify the top 5 hits (+)-tephrorin B, deguelin, vitamin p, lanceolarin and 3beta-hydroxy-20(29)-lupene with binding energy of -8.4, -8.1, -8.0, -7.8, and -7.8 kcal/mol, respectively. Furthermore, identified top 5 hits were subjected to drug-likeness and toxicity prediction as well as MM-GBSA calculation. Out of the five molecules four molecules were predicted not to comprise any mutagenic and carcinogenic effects. Top two molecules based on the drug-likeness properties for oral bio-availability were further analysed by molecular dynamics simulation at 100 ns timescale. It was observed from the dynamic behaviour of the two complexes that the addition of these molecules changed the conformation and stability of the apo protein; thus may act as inhibitors for Mpro.Communicated by Ramaswamy H. Sarma.

Enterocin LD3 from Enterococcus hirae LD3 Inhibits the Growth of Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311 in Fruit Juice.

In order to prevent the growth of pathogens in food, bacteriocins produced by various probiotic lactic acid bacteria have been recognized as potential substitutes of chemical preservatives. In this study, enterocin LD3 was purified from the cell-free supernatant of a food isolate, Enterococcus hirae LD3 using multistep chromatography. In the fruit juice, lethal concentration (LC50) of enterocin LD3 was found to be 260 µg/mL against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The cells treated with enterocin LD3 were red colour indicating dead cells after propidium iodide staining, while untreated cells were found blue after staining with 4', 6-diamidino-2-phenylindole. The mechanism of cell killing was analyzed using infrared spectrum of cells treated with enterocin LD3 which was found altered in the range of 1,094.30 and 1,451.82 cm-1 corresponding to nucleic acids and phospholipids, respectively. The morphology of target cells were severely ruptured and lysed as observed under electron microscopy. Thus, the present study suggested that enterocin LD3 showed bactericidal activity against Salm. enterica subsp. enterica serovar Typhimurium ATCC 13311 and may be applied as a bio-preservative for the safety of fruit juices.

A report of 5 Indian families with multicentric carpotarsal osteolysis syndrome.

Multicentric carpotarsal osteolysis syndrome (MCTO) is a rare autosomal dominant skeletal dysplasia characterised by swelling and restriction of movement in the wrist and ankle joints, as well as osteolysis of the carpal and tarsal bones, that can be misdiagnosed as juvenile idiopathic arthritis. We describe five Indian families with heterozygous nonrecurrent missense pathogenic variants in exon 1 of MAF bZIP transcription factor B (MAFB).

The exploitation of host autophagy and ubiquitin machinery by Mycobacterium tuberculosis in shaping immune responses and host defense during infection.

Intracellular pathogens have evolved various efficient molecular armaments to subvert innate defenses. Cellular ubiquitination, a normal physiological process to maintain homeostasis, is emerging one such exploited mechanism. Ubiquitin (Ub), a small protein modifier, is conjugated to diverse protein substrates to regulate many functions. Structurally diverse linkages of poly-Ub to target proteins allow enormous functional diversity with specificity being governed by evolutionarily conserved enzymes (E3-Ub ligases). The Ub-binding domain (UBD) and LC3-interacting region (LIR) are critical features of macroautophagy/autophagy receptors that recognize Ub-conjugated on protein substrates. Emerging evidence suggests that E3-Ub ligases unexpectedly protect against intracellular pathogens by tagging poly-Ub on their surfaces and targeting them to phagophores. Two E3-Ub ligases, PRKN and SMURF1, provide immunity against Mycobacterium tuberculosis (M. tb). Both enzymes conjugate K63 and K48-linked poly-Ub to M. tb for successful delivery to phagophores. Intriguingly, M. tb exploits virulence factors to effectively dampen host-directed autophagy utilizing diverse mechanisms. Autophagy receptors contain LIR-motifs that interact with conserved Atg8-family proteins to modulate phagophore biogenesis and fusion to the lysosome. Intracellular pathogens have evolved a vast repertoire of virulence effectors to subdue host-immunity via hijacking the host ubiquitination process. This review highlights the xenophagy-mediated clearance of M. tb involving host E3-Ub ligases and counter-strategy of autophagy inhibition by M. tb using virulence factors. The role of Ub-binding receptors and their mode of autophagy regulation is also explained. We also discuss the co-opting and utilization of the host Ub system by M. tb for its survival and virulence.Abbreviations: APC: anaphase promoting complex/cyclosome; ATG5: autophagy related 5; BCG: bacille Calmette-Guerin; C2: Ca2+-binding motif; CALCOCO2: calcium binding and coiled-coil domain 2; CUE: coupling of ubiquitin conjugation to ER degradation domains; DUB: deubiquitinating enzyme; GABARAP: GABA type A receptor-associated protein; HECT: homologous to the E6-AP carboxyl terminus; IBR: in-between-ring fingers; IFN: interferon; IL1B: interleukin 1 beta; KEAP1: kelch like ECH associated protein 1; LAMP1: lysosomal associated membrane protein 1; LGALS: galectin; LIR: LC3-interacting region; MAPK11/p38: mitogen-activated protein kinase 11; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAPK8/JNK: mitogen-activated protein kinase 8; MHC-II: major histocompatibility complex-II; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; NFKB1/p50: nuclear factor kappa B subunit 1; OPTN: optineurin; PB1: phox and bem 1; PE/PPE: proline-glutamic acid/proline-proline-glutamic acid; PknG: serine/threonine-protein kinase PknG; PRKN: parkin RBR E3 ubiquitin protein ligase; RBR: RING-in between RING; RING: really interesting new gene; RNF166: RING finger protein 166; ROS: reactive oxygen species; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6; Ub: ubiquitin; UBA: ubiquitin-associated; UBAN: ubiquitin-binding domain in ABIN proteins and NEMO; UBD: ubiquitin-binding domain; UBL: ubiquitin-like; ULK1: unc-51 like autophagy activating kinase 1.

Diagnostic and prognostic biomarkers in colorectal cancer and the potential role of exosomes in drug delivery.

Colorectal cancer (CRC) is the third most common cancer with the second most frequent cause of death worldwide. One fourth to one fifth of the CRC cases are detected at advance stage. Early detection of colorectal cancer might help in decreasing mortality and morbidity worldwide. CRC being a heterogeneous disease, new non-invasive approaches are needed to complement and improve the screening and management of CRC. Reliable and early detectable biomarkers would improve diagnosis, prognosis, therapeutic responses, and will enable the prediction of drug response and recurrence risk. Over the past decades molecular research has demonstrated the potentials of CTCs, ctDNAs, circulating mRNAs, ncRNAs, and exosomes as tumor biomarkers. Non-invasive screening approaches using fecal samples for identification of altered gut microbes in CRC is also gaining attention. Exosomes can be potential candidates that can be employed in the drug delivery system. Further, the integration of in vitro, in vivo and in silico models that involve CRC biomarkers will help to understand the interactions occurring at the cellular level. This review summarizes recent update on CRC biomarkers and their application along with the nanoparticles followed by the application of organoid culture in CRC.

Chest wall metastasis of squamous cell carcinoma of larynx.

Distant metastases in squamous cell carcinoma of head and neck are most often to the lung, liver and bone. They rarely metastasise to chest wall. We report a 60-year-old male patient who initially presented with an abscess over the anterior chest wall that was initially treated for infective pathology. Due to lack of response, cytological examination was performed that turned out to be metastasis from carcinoma larynx.

Honeybee nutrition and pollen substitutes: A review.

Like other invertebrates, honey bees too are poikilothermic animals; they cannot regulate their body temperature and they have to undergo a period of inactivation when atmospheric temperature is un-tolerable. During this period, their nutritional requirements and metabolic activities are minimized due to highly restricted foraging activities. The egg-laying by queen and rearing of unsealed and sealed brood are decreased, however their extent is governed by the quantum of stored food available. The problems of deleterious influence of adverse weather conditions and non-availability of bee flora all round the year, in a particular locality, have been realized by the researchers/beekeepers and migration concept has been developed to solve this problem. But again, migration itself is not an easy task. The provision of artificial feeding as an alternate of migration. Scientists all over the world have formulated different artificial food recepies for bees on the basis of nutrient composition of honey and pollen, acceptability, palatability, digestibility and affordability of ingredients. This may help to maintain all colony parameters enough to derive maximum advantage of forthcoming floral rich season. However, a standard balanced diet for commercial beekeeping that is accepted worldwide is still awaited.

Mycobacterium tuberculosis Specific Protein Rv1509 Evokes Efficient Innate and Adaptive Immune Response Indicative of Protective Th1 Immune Signature.

Dissecting the function(s) of proteins present exclusively in Mycobacterium tuberculosis (M.tb) will provide important clues regarding the role of these proteins in mycobacterial pathogenesis. Using extensive computational approaches, we shortlisted ORFs/proteins unique to M.tb among 13 different species of mycobacteria and identified a hypothetical protein Rv1509 as a 'signature protein' of M.tb. This unique protein was found to be present only in M.tb and absent in all other mycobacterial species, including BCG. In silico analysis identified numerous putative T cell and B cell epitopes in Rv1509. Initial in vitro experiments using innate immune cells demonstrated Rv1509 to be immunogenic with potential to modulate innate immune responses. Macrophages treated with Rv1509 exhibited higher activation status along with substantial release of pro-inflammatory cytokines. Besides, Rv1509 protein boosts dendritic cell maturation by increasing the expression of activation markers such as CD80, HLA-DR and decreasing DC-SIGN expression and this interaction was mediated by innate immune receptor TLR2. Further, in vivo experiments in mice demonstrated that Rv1509 protein promotes the expansion of multifunctional CD4+ and CD8+T cells and induces effector memory response along with evoking a canonical Th1 type of immune response. Rv1509 also induces substantial B cell response as revealed by increased IgG reactivity in sera of immunized animals. This allowed us to demonstrate the diagnostic efficacy of this protein in sera of human TB patients compared to the healthy controls. Taken together, our results reveal that Rv1509 signature protein has immunomodulatory functions evoking immunological memory response with possible implications in serodiagnosis and TB vaccine development.

MycoVarP: Mycobacterium Variant and Drug Resistance Prediction Pipeline for Whole-Genome Sequence Data Analysis.

Whole-genome sequencing (WGS) provides a comprehensive tool to analyze the bacterial genomes for genotype-phenotype correlations, diversity of single-nucleotide variant (SNV), and their evolution and transmission. Several online pipelines and standalone tools are available for WGS analysis of Mycobacterium tuberculosis (Mtb) complex (MTBC). While they facilitate the processing of WGS data with minimal user expertise, they are either too general, providing little insights into bacterium-specific issues such as gene variations, INDEL/synonymous/PE-PPE (IDP family), and drug resistance from sample data, or are limited to specific objectives, such as drug resistance. It is understood that drug resistance and lineage-specific issues require an elaborate prioritization of identified variants to choose the best target for subsequent therapeutic intervention. Mycobacterium variant pipeline (MycoVarP) addresses these specific issues with a flexible battery of user-defined and default filters. It provides an end-to-end solution for WGS analysis of Mtb variants from the raw reads and performs two quality checks, viz, before trimming and after alignments of reads to the reference genome. MycoVarP maps the annotated variants to the drug-susceptible (DS) database and removes the false-positive variants, provides lineage identification, and predicts potential drug resistance. We have re-analyzed the WGS data reported by Advani et al. (2019) using MycoVarP and identified some additional variants not reported so far. We conclude that MycoVarP will help in identifying nonsynonymous, true-positive, drug resistance-associated variants more effectively and comprehensively, including those within the IDP of the PE-PPE/PGRS family, than possible from the currently available pipelines.

Mutually exclusive locales for N-linked glycans and disorder in human glycoproteins.

Several post-translational protein modifications lie predominantly within regions of disorder: the biased localization has been proposed to expand the binding versatility of disordered regions. However, investigating a representative dataset of 500 human N-glycoproteins, we observed the sites of N-linked glycosylations or N-glycosites, to be predominantly present in the regions of predicted order. When compared with disordered stretches, ordered regions were not found to be enriched for asparagines, serines and threonines, residues that constitute the sequon signature for conjugation of N-glycans. We then investigated the basis of mutual exclusivity between disorder and N-glycosites on the basis of amino acid distribution: when compared with control ordered residue stretches without any N-glycosites, residue neighborhoods surrounding N-glycosites showed a depletion of bulky, hydrophobic and disorder-promoting amino acids and an enrichment for flexible and accessible residues that are frequently found in coiled structures. When compared with control disordered residue stretches without any N-glycosites, N-glycosite neighborhoods were depleted of charged, polar, hydrophobic and flexible residues and enriched for aromatic, accessible and order-promoting residues with a tendency to be part of coiled and ß structures. N-glycosite neighborhoods also showed greater phylogenetic conservation among amniotes, compared with control ordered regions, which in turn were more conserved than disordered control regions. Our results lead us to propose that unique primary structural compositions and differential propensities for evolvability allowed for the mutual spatial exclusion of N-glycosite neighborhoods and disordered stretches.

Author Correction: Mutually exclusive locales for N-linked glycans and disorder in human glycoproteins.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nucleotide conjugated (ZnO)3 cluster: Interaction and optical characteristics using TDDFT.

Binding of four DNA nucleotide units with (ZnO)3 cluster in an aqueous phase has been investigated using density functional theory (DFT) and time dependent-density functional theory (TDDFT) method and the stability order for (ZnO)3-nucleobases/sugar/phosphate systems is predicted as phosphate > C > A > S > T ∼ G. The order of binding energy for (ZnO)3-nucleotide hybrid systems is observed to be (ZnO)3 + nuc-C ˃ (ZnO)3 + nuc-A ˃ (ZnO)3 + nuc-G ˃ (ZnO)3 + nuc-T. The binding of nucleotide units with the cluster has been explained on the basis of molecular electrostatic potential (MEP) plots, hydrogen bonding, glycosidic torsion angles, density of state (DOS) plots. The photophysical properties of (ZnO)3-nucleotide complexes have been studied using TDDFT approach. Among all (ZnO)3-nucleotide complexes, the absorption spectra of (ZnO)3 + nuc-A and (ZnO)3 + nuc-C complexes are seen to undergo red shift with respect to their bare nucleotide units that would be useful in the optical sensing of the respective nucleotides of DNA. It is interesting to note that binding of the nucleotide unit with the cluster makes it fluorescent, the study reports the fluorescence activity of (ZnO)3 + nuc-T complex.

A QM/MM study on ethene and benzene oxidation using silica-supported chromium trioxide.

Oxidation of ethene and benzene by chromium oxide (CrO3) supported on silica (SiO2) was investigated by employing hybrid quantum mechanics/molecular mechanics (QM/MM) model calculations. Various mechanistic possibilities, such as C-H or C=C bond activation of hydrocarbons, were investigated in detail for the reaction of ethene and benzene with CrO3 grafted on a silica surface. While activation of the C-H bond leads to the formation of alcohol, epoxide is obtained via C=C bond activation. The complete reaction routes for the formation of each product were traced and found to be exothermic. Thermochemical analysis were performed to predict temperature conditions for the reaction to be feasible in a forward direction. The study provides conclusive evidence to aid experimentalists for further research on oxidation of hydrocarbons using silica-supported metal oxides. Graphical abstract Oxidation of ethene and benzene using silica-supported chromium trioxide.

Evolution of catalytic microenvironment governs substrate and product diversity in trichodiene synthase and other terpene fold enzymes.

Trichodiene synthase, a terpene fold enzyme catalyzes the first reaction of trichodermin biosynthesis that is an economically important secondary metabolite. Sequence search analysis revealed that the proteins containing terpene fold are present in bacteria, fungi and plants. Terpene fold protein from Selaginella moellendorffii, a lycophyte, appeared at the interface of the microbes and plants in the evolutionary scale. Amino acid residues present around the catalytic pocket determines the size of the substrate as well as product molecules. It has been observed that the overall molecular evolution of the catalytic pockets dictates the choice of substrates/products of the proteins. It was further observed that N-terminus of multi-domain terpene fold proteins may assist in the interactions with the pyrophosphate part of the substrates. The phylogenetic analysis of these proteins further revealed that the enzymes are clustered into groups based on the domains present additional to the catalytic domains. We have also observed inter-domain 'puckering forceps' type motions in the multi-domains using normal mode analysis which were further correlated with their functions. The evolutionary clustering of these proteins was also influenced by the presence/absence of cofactor interacting motifs. These results may be used to modify/enhance the functions of these enzymes using protein engineering methods.