International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 1(6), 38-42, October (2012) I. Res. J. Biological Sci. International Science Congress Association 38 Insights to Sequence Information of Lactoylglutathione Lyase Enzyme from Different Source OrganismsDwivedi Vivek Dhar¹, Sharma Tanuj¹, Mishra Sarad Kumar² and Pandey Amit Kumar³ ¹Department of Bioinformatics, UCST, Dehradun, INDIA ²Department of Biotechnology, DDU University, Gorakhpur, INDIA ³Department of Forest Pathology, FRI, Dehradun, INDIAAvailable online at: www.isca.in Received 25th July 2012, revised 4th August 2012, accepted 14th August 2012Abstract Lactoylglutathione lyases (also known as glyoxalase I) are widely distributed enzymes among plants, fungi and bacteria. It is an enzyme that catalyzes the isomerization of hemithioacetal adducts, which are formed in a spontaneous reaction between a glutathionyl group and aldehydes such as methylglyoxal. In the present study, thirty full- length amino acid sequences of lactoylglutathione lyases from bacteria, fungi, and plants were collected and subjected to multiple sequence alignment (MSA), pattern identification, domain identification discovering individual amino acid composition, and phylogenetic tree construction. MSA revealed that one tyrosine residue were identically found in all analyzed species, two tyrosine, one arginine, one leucine, one glycine, one histidine, one phenylalanine, one proline, one aspartic acid and one glutamic acid residues were identically found in all the bacterial and fungal sources, one phenylalanine, one tyrosine, one histidine, one proline, and one glycine residues were identically found in all bacterial and plant sources while two glycine, two tyrosine, two aspartic acid and one proline residues were identically found in all plants and fungal lactoylglutathione lyases. Two major sequence clusters were constructed by phylogenetic analysis. One cluster contains ten species of fungi, five species of plant, and two species of bacteria, whereas the other one contains eight species of bacteria, four species of plant and one species of plant was outgrouped from both clusters . The amino acid composition result revealed that the average frequency of amino acid glycine is 7.86 percent that is very high in comparison to other amino acids and an average frequency of is 1.07 that is very low in all analyzed species. In addition, nine motifs which were unique for their groups were also identified. Keywords: Lactoylglutathione lyase, phylogenetic analysis, conserved regions, motifs, Domains, amino acid composition. IntroductionLactoylglutathione lyase (LGL) is an enzyme involved in the detoxification of methylglyoxal, a highly toxic electrophilic glycolytic by-product that reacts with and inactivates intracellular macromolecules, including both proteins and nucleic acids. Therefore, its rapid degradation is vital for cell survival. The formation of methylglyoxal occurs via enzymatic production during glycolysis from the fragmentation of triose phosphates2-7. LGL is involved in methylglyoxal detoxification via the formation of -d-lactoylglutathione from the hemimercaptal adduct that is formed nonenzymatically between glutathione and the 2-oxoaldehyde methylglyoxal. Glyoxalase II then converts -d-lactoylglutathione into reduced glutathione and d-lactate8-11. An examination of the S. mutans UA159 genome does not reveal the presence of a glyoxalase II homologue, suggesting an alternate pathway by which -d-lactoylglutathione is neutralized. In Escherichia coli, methylglyoxal was accumulated under physiological conditions of uncontrolled carbohydrate metabolism and the concentration of methylglyoxal was seen to be greater in highly metabolically active human red blood cells, where lgl expression appeared to be regulated by the rate of glycolysis12-13. Considering the above facts, a study of amino acid sequences of lactoylglutathione lyase from different sources of organisms is quite challenging. In the present study, we performed the individual in silicostudies of amino acid sequences obtained from bacteria, fungi, and plants, and correlated them on the basis of some common features. Material and Methods The full-length amino acid sequences of lactoylglutathione lyase from bacteria, fungi and plants were searched and retrieved from ntrez protein database available at NCBI. The sequences were arranged in bacterial, fungal, and plant profile, respectively. The multiple sequence alignment of the individual profiles was performed using MUSCLE at the European Bioinformatics Institute. Motifs were discovered in profiles using the expectation maximization approach14 implemented in ultiple EM for otif licitation server. Further, the discovered motifs were used to search their protein family using Pfam at the anger nstitute. The UPGMA approach implemented in the ega program was employed for constructing phylogenetic relationships among sequences. The statistical reliability of the phylogenetic tree was tested by bootstrap analyses with 500 replications. Mega program is also used for discovering individual amino acid composition. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(6), 38-42, October (2012) I. Res. J. Biological Sci. International Science Congress Association 39 Results and DiscussionThe accession number of retrieved sequences along with the species name and origin is listed in able- 1 . For MSA four profiles were created. One is from bacterial and fungal origin, second is from bacterial and plants origin, third is from lants and ungal origin, and fourth is from all bacterial, fungal, and plants origin. MSA showed the presence of some conserved regions in all the sequences from different profiles, while others were restricted only to their groups. one tyrosine residue were identically found in all analyzed species, two tyrosine, one arginine, one leucine, one glycine, one histidine, one phenylalanine, one proline, one aspartic acid and one glutamic acid residues were identically found in all the bacterial and fungal sources, one phenylalanine, one tyrosine, one histidine, one proline, and one glycine residues were identically found in all bacterial and plant sources while two glycine, two tyrosine, two aspartic acid and one proline residues were identically found in all plant and fungal lactoylglutathione lyase. For phylogenetic analysis four major profiles were created. Profile one was of bacterial origin, second was of fungal origin, third was of plants origin, and fourth was joint profile of bacterial, fungal, and plants, origin. Phylogenetic analysis15 of bacterial profile showed two major clusters (figure-1). Cluster I consist of seven species which was further divided into two subclusters. Subcluster I contains four species (Ferrimonas balearica, Mesorhizobium amorphae,Francisella noatunensis, and Mesorhizobium opportunistum). Subcluster II contains three species (Marinobacter adhaerens,Cardiobacterium valvarum, and Halomonas sp.). Cluster II consist of three species namely Agrobacterium tumefaciens, Clostridium perfringens, and Streptococcus pyogenes. Figure-1 Phylogenetic analysis of bacterial profile using UPGMA method Phylogenetic analysis16 of fungal profile showed two major clusters (figure-2). Cluster I consist of four species which was further divided into two subclusters. Subcluster I contains two species (Metarhizium acridum, and Magnaporthe oryzae). Subcluster II also contains two species (Neurospora tetrasperma, and Neurospora crassa). Cluster II consist of two species namely Colletotrichum higginsianum, and Glomerella graminicola. Verticillium dahliae,Exophiala dermatitidis, Paracoccidioides brasiliensis and Metarhizium anisopliae were ditantantly related to each other and not included in any cluster. Table -1 Retrieved sequence from NCBI/Entrez and their accession number Source Species Accession no. Bacteria Ferrimonas balearica YP_003913279.1 Bacteria Marinobacter adhaerens ADP98654.1 Bacteria Mesorhizobium opportunistum AEH89136.1 Bacteria Francisella noatunensis AFJ43975.1 Bacteria Clostridium perfringens EIA18179.1 Bacteria Streptococcus pyogenes NP_268789.1 Bacteria Cardiobacterium valvarum ZP_09445317.1 Bacteria Mesorhizobium amorphae ZP_09089707.1 Bacteria Agrobacterium tumefaciens EHH07353.1 Bacteria Halomonas sp. ZP_08960109.1 Fungi Neurospora tetrasperma EGZ68442.1 Fungi Metarhizium anisopliae EFY94694.1 Fungi Metarhizium acridum EFY91247.1 Fungi Magnaporthe oryzae EHA48833.1 Fungi Colletotrichum higginsianum CCF47148.1 Fungi Exophiala dermatitidis EHY60225.1 Fungi Verticillium dahliae EGY14417.1 Fungi Paracoccidioides brasiliensis EEH40065.1 Fungi Glomerella graminicola EFQ30294.1 Fungi Neurospora crassa XP_960441.1 Plant Zea mays NP_001146873.1 Plant Arabidopsis thaliana AEE28246.1 Plant Medicago truncatula AES92643.1 Plant Vitis vinifera XP_002273346.2 Plant Oryza sativa AEK99333.1 Plant Cucumis melo ADN34057.1 Plant Gossypium hirsutum ACJ11750.1 Plant Glycine max NP_001236152.1 Plant Solanum lycopersicum NP_001234447.1 Plant Arachis hypogaea ACF74334.1 Ferrimonas balearica Mesorhizobium amorphae Francisella noatunensis Mesorhizobium opportunistum Marinobacter adhaerens Cardiobacterium valvarum Halomonas sp. Agrobacterium tumefaciens Clostridium perfringens Streptococcus pyogenes 86969679559958 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(6), 38-42, October (2012) I. Res. J. Biological Sci. International Science Congress Association 40 Figure-2 Phylogenetic analysis of fungal profile using UPGMA method Figure-3 Phylogenetic analysis of plants profile using UPGMA method Phylogenetic analysis of plant profile showed two major clusters (figure-3). Cluster I consist of five species which was further divided into two subclusters. Subcluster I contains three species Glycine max,Solanum lycopersicum, and Arabidopsis thaliana). Subcluster II also contains two species (Zea mays, and Oryza sativa). Cluster II consist of four species namely Cucumis melo, Medicago truncatula, Vitis vinifera and Gossypium hirsutum. Arachis hypogaea was distantly related and not included in any cluster. When joint profile of bacteria, fungi, and plant, sequences were taken for phylogenetic analysis, two major clusters were formed igure-4). One cluster contains ten species of fungi, five species of plant, and two species of bacteria, whereas the other one contains eight species of bacteria, four species of plant and one species of plant was outgrouped from both clusters . This suggests that fungi were closely related to plants in comparison to bacteria in terms of lactoylglutathione lyases . Nine motifs are identified with their Pfam analysis which was unique for their groups given in able- 2 . The amino acid composition result revealed that the average frequency of amino acid glycine is 7.86 percent that is very high in comparison to other amino acids and an average frequency of is 1.07 that is very low in all analyzed species igure-4) . Figure-4 Phylogenetic analysis of joint profile of bacterial, fungal, and plants sequences using UPGMA method Metarhizium acridum Magnaporthe oryzae Neurospora tetrasperma Neurospora crassa Colletotrichum higginsianum Glomerella graminicola Verticillium dahliae Exophiala dermatitidis Paracoccidioides brasiliensis Metarhizium anisopliae 1005148964148100 Glycine max Solanum lycopersicum Arabidopsis thaliana Zea mays Oryza sativa Cucumis melo Medicago truncatula Vitis vinifera Gossypium hirsutum Arachis hypogaea 99717010010090100 Metarhizium acridum Magnaporthe oryzae Neurospora tetrasperma Neurospora crassa Colletotrichum higginsianum Glomerella graminicola Verticillium dahliae Exophiala dermatitidis Paracoccidioides brasiliensis Metarhizium anisopliae Cardiobacterium valvarum Halomonas sp. Marinobacter adhaerens Zea mays Oryza sativa Arabidopsis thaliana Glycine max Solanum lycopersicum Mesorhizobium opportunistum Agrobacterium tumefaciens Clostridium perfringens Streptococcus pyogenes Ferrimonas balearica Mesorhizobium amorphae Francisella noatunensis Cucumis melo Medicago truncatula Vitis vinifera Gossypium hirsutum Arachis hypogaea 9965100100994231815870999488489067333289739382100871005048 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(6), 38-42, October (2012) I. Res. J. Biological Sci. International Science Congress Association 41 Table-2 Motifs searched with meme program Source Motif widthMotif present in number of sequences Motif Pfam Bacteria 36 9 MKFLHTMLRVKDLDRSLDFYTNAFGMTEVRRLDFEE Glyoxalase family Bacteria 16 9 IAFIKDPDGYKIEVIQ Glyoxalase 2 family Bacteria 21 9 YDEGNGFGHIAVGVEDIYAAC Pfam entry not found Fungi 50 8 PQGFGHICVSVDDIDAACARFEALKVNWKKRLTDGRMKNVAFLLDPDNYW Glyoxalase 2 family Fungi 50 8 IELTHNYGTENDPSYTVNNGNTEPHRGFGHTCISVDNIQAACQRLEDAGY Glyoxalase 4 family Fungi 41 8 HSMIRVKDPKASVKFYELLGMSVIKKLEFPEAKFDLYFLAY Glyoxalase family Plant 41 10 QQTMLRVKDPKRSLDFYSKVLGMSLLKRLDFPEMKFSLYFM Glyoxalase family Plant 21 10 RGFGHIGVTVDDVYKACERFE Glyoxalase family Plant 41 10 APVDDLDRTIWTFTQCATMELTHNWGTEEDPEFKGYHNGNS Pfam entry not found Figure-5 Amino acid composition of lactoylglutathione lyase (given in percent) in different source organisms International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(6), 38-42, October (2012) I. Res. J. Biological Sci. International Science Congress Association 42 Conclusion In silico analysis of the sequences showed sequence based similarities depending on their source organism. one tyrosine residue were identically found in all analyzed species, two tyrosine, one arginine, one leucine, one glycine, one histidine, one phenylalanine, one proline, one aspartic acid and one glutamic acid residues were identically found in all the bacterial and fungal sources, one phenylalanine, one tyrosine, one histidine, one proline, and one glycine residues were identically found in all bacterial and plant sources while two glycine, two tyrosine, two aspartic acid and one proline residues were identically found in all plant and fungal lactoylglutathione lyase. This suggests that these conserved amino acid residues have an important function in lactoylglutathione lyase sequences and in its evolution from lower organisms (bacteria) to higher organisms (plants). Some motifs which were unique for their group were also identified. In all species of bacteria, fungi, and plants an average frequency of amino acid glycine is 7.86 percent that is very high in comparison to other amino acids. This suggests that the amino acid glycine play a very important role in the composition lactoylglutathione lyases. Two major sequence clusters were constructed by phylogenetic analysis One cluster contains ten species of fungi, five species of plant, and two species of bacteria, whereas the other one contains eight species of bacteria, four species of plant and one species of plant was outgrouped from both clusters . This classification can significantly contribute in the understanding of the evolutionary relations between the species at molecular level. However, owing to the considerable importance of lactoylglutathione lyase, more contribution is warranted for the detailed investigation of the activity and functional analysis of enzymes. AcknowledgementsWe are thankful to department of biotechnology DDU Gorakhpur University, Gorakhpur for providing laboratory facilities & encouragement and to director of UCST, Dehradun for their cooperation during the study. We are grateful to head of Department of Forest Pathology Division, FRI, Dehradun for his kind support and necessary suggestions whenever I needed. References 1.Ackerman R.S., Cozzarelli N.R. and Epstein W., Accumulation of toxic concentrations of methylglyoxal by wild-type Escherichia coli K-12, J. Bacteriol, 119, 357-362 (1974) 2.Allen R.E., Lo T.W. and Thornalley P.J., Purification and characterisation of glyoxalase II from human red blood cells, Eur. J. Biochem., 213, 1261-1267 (1993) 3.Banas J.A., Virulence properties of Streptococcus mutans, Front. Biosci., 9, 1267-1277 (2004) 4.Cooper R.A., Metabolism of methylglyoxal in microorganisms, Annu. Rev. Microbiol., 38, 49-68 (1984) 5.Eriksson S., Lucchini S., Thompson A., Rhen M., and Hinton J.C., Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica, Mol. Microbiol, 47, 103-118 (2003) 6.Freedberg W.B., Kistler W.S. and Lin E.C., Lethal synthesis of methylglyoxal by Escherichia coli during unregulated glycerol metabolism, J. Bacteriol, 108, 137-144 (1971) 7.Frickel E.M., Jemth P., Widersten M. and Mannervik B., Yeast glyoxalase I is a monomeric enzyme with two active sites, J. Biol. Chem., 276, 1845-1849 (2001) 8.Hanna M.N., Ferguson R.J., Li Y.H. and Cvitkovitch D.G., uvrA is an acid-inducible gene involved in the adaptive response to low pH in Streptococcus mutans, J. Bacteriol., 183, 5964-5973 (2001)9.Inoue Y. and Kimura A., Methylglyoxal and regulation of its metabolism in microorganisms, Adv. Microb. Physiol., 37, 177-227 (1995) 10.Kalapos M.P., Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications, Toxicol. Lett., 110, 145-175 (1999) 11.Khalichi P., Cvitkovitch D.G. and Santerre J.P., Effect of composite resin biodegradation products on oral streptococcal growth, Biomaterials,25, 5467-5472 (2004)12.Kim I., Kim E., Yoo S., Shin D., Min B., Song J. and Park C., Ribose utilization with an excess of mutarotase causes cell death due to accumulation of methylglyoxal, J. Bacteriol., 186, 7229-7235 (2004) 13.Ko J., Kim I., Yoo S., Min B., Kim K. and Park C., Conversion of methylglyoxal to acetol by Escherichia colialdo-keto reductases, J. Bacteriol., 187, 5782-5789 (2005)14.Bailey T.L. and Elkan C., Expectation maximization pproach, Mach Learn, 21(1–2), 51–80 (1995)15.Dwivedi V.D. and Mishra S.K., Amino acid sequence analysis of glutamate dehydrogenase from different source organisms, Online J Bioinform., 13(2), 184-191 (2012)16.Bhatt T.K., Phylogenetic Studies on tRNA Dependent Amidotransferase from Plasmodium Falciparum,ISCA J. Biological Sci., 1(3), 20-24 (2012)