Abstract
Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys–His–Asp and a G–x–C–x–G–G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni–NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90% of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5–9 when incubated for 60 min (more than 50% residual activity). Its Km value was 256 μM and its catalytic efficiency was 81.7 s−1. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein.
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References
Alias N, Mazian A, Salleh AB et al (2014) Molecular cloning and optimization for high level expression of cold-adapted serine protease from antarctic yeast Glaciozyma antarctica PI12. Enzyme Res 2014 (article ID 197938). https://doi.org/10.1155/2014/197938
Alvarez M, Zeelen JP, Mainfroid V et al (1998) Triose-phosphate isomerase (TIM) of the psychrophilic bacterium Vibrio marinus kinetic and structural properties. J Biol Chem 273:2199–2206. https://doi.org/10.1074/jbc.273.4.2199
Amato P, Christner BC (2009) Energy metabolism response to low-temperature and frozen conditions in Psychrobacter cryohalolentis. Appl Environ Microbiol 75:711–718. https://doi.org/10.1128/AEM.02193-08
Bateman A, Coin L, Durbin R et al (2004) The Pfam protein families database. Nucleic Acids Res 32:D138–D141. https://doi.org/10.1093/nar/gkh121
Bharudin I, Zaki NZ, Bakar FDA et al (2014) Comparison of RNA extraction methods for transcript analysis from the psychrophilic yeast, Glaciozyma antarctica. Malays Appl Biol 43:71–79
Boo SY, Wong CMVL, Rodrigues KF et al (2013) Thermal stress responses in antarctic yeast, Glaciozyma antarctica PI12, characterized by real-time quantitative PCR. Polar Biol 36:381–389. https://doi.org/10.1007/s00300-012-1268-2
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Cámara B, Marín M, Schlömann M et al (2008) trans-Dienelactone hydrolase from Pseudomonas reinekei MT1, a novel zinc-dependent hydrolase. Biochem Biophys Res Commun 376:423–428. https://doi.org/10.1016/j.bbrc.2008.09.006
Cavicchioli R, Siddiqui KS, Andrews D et al (2002) Low-temperature extremophiles and their applications. Curr Opin Biotechnol 13:253–261. https://doi.org/10.1016/S0958-1669(02)00317-8
Cavicchioli R, Charlton T, Ertan H et al (2011) Biotechnological uses of enzymes from psychrophiles. Microb Biotechnol 4:449–460. https://doi.org/10.1111/j.1751-7915.2011.00258.x
Costantini S, Colonna G, Facchiano AM (2008) ESBRI: a web server for evaluating salt bridges in proteins. Bioinformation 3:137–138. https://doi.org/10.6026/97320630003137
DasSarma S, Capes MD, Karan R et al (2013) Amino acid substitutions in cold-adapted proteins from Halorubrum lacusprofundi, an extremely halophilic microbe from Antarctica. PLoS ONE 8:e58587. https://doi.org/10.1371/journal.pone.0058587
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. https://doi.org/10.1093/nar/gkh340
Feller G (2013) Psychrophilic enzymes: from folding to function and biotechnology. Scientifica 2013 (article ID 512840). https://doi.org/10.1155/2013/512840
Feller G, Gerday C (2003) Psychrophilic enzymes: hot topics in cold adaptation. Nat Rev Microbiol 1:200–208. https://doi.org/10.1038/nrmicro773
Fretwell P, Pritchard HD, Vaughan DG et al (2013) Bedmap 2: improved ice bed, surface and thickness datasets for Antarctica. Cryosphere 7:375–393. https://doi.org/10.5194/tc-7-375-2013
Gerday C, Aittaleb M, Bentahir M et al (2000) Cold-adapted enzymes: from fundamentals to biotechnology. Trends Biotechnol 18:103–107. https://doi.org/10.1016/S0167-7799(99)01413-4
Hashim NHF, Bharudin I, Nguong DLS et al (2013) Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12. Extremophiles 17:63–73. https://doi.org/10.1007/s00792-012-0494-4
Hunter S, Apweiler R, Attwood TK et al (2009) Interpro: the integrative protein signature database. Nucleic Acids Res 37:D211–D215. https://doi.org/10.1093/nar/gkn785
Huston AL, Haeggström JZ, Feller G (2008) Cold adaptation of enzymes: structural, kinetic and microcalorimetric characterizations of an aminopeptidase from the Arctic psychrophile Colwellia psychrerythraea and of human leukotriene A4 hydrolase. Biochim Biophys Acta 1784:1865–1872. https://doi.org/10.1016/j.bbapap.2008.06.002
Kumar A, Pillay B, Olaniran AO (2014) Two structurally different dienelactone hydrolases (TfdeI and TfdeII) from Cupriavidus necator JMP134 plasmid pJP4 catalyse cis- and trans-dienelactones with similar efficiency. PLoS ONE 9:e101801. https://doi.org/10.1371/journal.pone.0101801
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Leiros I, Moe E, Lanes O et al (2003) The structure of uracil-DNA glycosylase from Atlantic cod (Gadus morhua) reveals cold-adaptation features. Acta Cryst D59:1357–1365. https://doi.org/10.1107/S0907444903011144
Leiros H-KS, Pey AL, Innselset M et al (2007) Structure of phenylalanine hydroxylase from Colwellia psychrerythraea 34H, a monomeric cold active enzyme with local flexibility around the active site and high overall stability. J Biol Chem 282:21973–21986. https://doi.org/10.1074/jbc.M610174200
Marín M, Pieper DH (2009) Novel metal-binding site of Pseudomonas reinekei MT1 trans-dienelactone hydrolase. Biochem Biophys Res Commun 390:1345–1348. https://doi.org/10.1016/j.bbrc.2009.10.151
Marx J-C, Collins T, D’Amico S et al (2007) Cold-adapted enzymes from marine Antarctic microorganisms. Mar Biotechnol 9:293–304. https://doi.org/10.1007/s10126-006-6103-8
Moiseeva OV, Solyanikova IP, Kaschabek SR et al (2002) A new modified ortho cleavage pathway of 3-chlorocatechol degradation by Rhodococcus opacus 1CP: genetic and biochemical evidence. J Bacteriol 184:5282–5292. https://doi.org/10.1128/JB.184.19.5282-5292.2002
Nardini M, Dijkstra BW (1999) α/β hydrolase fold enzymes: the family keeps growing. Curr Opin Struct Biol 9:732–737. https://doi.org/10.1016/S0959-440X(99)00037-8
Park S-Y, Kim J-T, Kang SG et al (2007) A new esterase showing similarity to putative dienelactone hydrolase from a strict marine bacterium, Vibrio sp. Gmd509. Appl Microbiol Biot 77:107–115. https://doi.org/10.1007/s00253-007-1134-2
Park Y-J, Yoon S-J, Lee H-B (2010) A novel dienelactone hydrolase from the thermoacidophilic archaeon Sulfolobus solfataricus P1: purification, characterization, and expression. Biochim Biophys Acta 1800:1164–1172. https://doi.org/10.1016/j.bbagen.2010.07.006
Pathak D, Ollis D (1990) Refined structure of dienelactone hydrolase at 1.8 Å. J Mol Biol 214:497–525. https://doi.org/10.1016/0022-2836(90)90196-S
Pettersen EF, Goddard TD, Huang CC et al (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612. https://doi.org/10.1002/jcc.20084
Priscu JC, Christner BC (2004) Earth’s icy biosphere. In: Bull A (ed) Microbial diversity and bioprospecting. ASM Press, Washington, pp 130–145. https://doi.org/10.1128/9781555817770.ch13
Ramli ANM, Mahadi NM, Rabu A et al (2011) Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12. Microb Cell Fact 10:94. https://doi.org/10.1186/1475-2859-10-94
Ramli ANM, Mahadi NM, Shamsir MS et al (2012) Structural prediction of a novel chitinase from the psychrophilic Glaciozyma antarctica PI12 and an analysis of its structural properties and function. J Comput Aided Mol Des 26:947–961. https://doi.org/10.1007/s10822-012-9585-7
Schlömann M (1994) Evolution of chlorocatechol catabolic pathways. Conclusions to be drawn from comparisons of lactone hydrolases. Biodegradation 5:301–321. https://doi.org/10.1007/BF00696467
Siddiqui KS, Cavicchioli R (2006) Cold-adapted enzymes. Annu Rev Biochem 75:403–433. https://doi.org/10.1146/annurev.biochem.75.103004.142723
Siddiqui KS, Williams TJ, Wilkins D et al (2013) Psychrophiles. Annu Rev Earth Planet Sci 41:87–115. https://doi.org/10.1146/annurev-earth-040610-133514
Söding J, Biegert A, Lupas AN (2005) The HHPred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 33:W244–W248. https://doi.org/10.1093/nar/gki408
Tamura K, Dudley J, Nei M et al (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599. https://doi.org/10.1093/molbev/msm092
Thomas DN, Dieckmann GS (2002) Antarctic sea ice—a habitat for extremophiles. Science 295:641–644. https://doi.org/10.1126/science.1063391
Tina KG, Bhadra R, Srinivasan N (2007) PIC: protein interactions calculator. Nucleic Acids Res 35:W473–W476. https://doi.org/10.1093/nar/gkm423
Violot S, Aghajari N, Czjzek M et al (2005) Structure of a full length psychrophilic cellulase from Pseudoalteromonas haloplanktis revealed by X-ray diffraction and small angle X-ray scattering. J Mol Biol 348:1211–1224. https://doi.org/10.1016/j.jmb.2005.03.026
Webb B, Sali A (2014) Comparative protein structure modeling using MODELLER. Curr Protoc Bioinform 47:561–5632. https://doi.org/10.1002/0471250953.bi0506s15
Acknowledgements
We would like to thank the Ministry of Science, Technology and Innovation (MOSTI), Malaysia for funding this research under the Grant SF 02-05-20-SF0007. We acknowledge the support given by the Australian Antarctic Division and the Malaysian Antarctic Research Program (MARP) of the Academy of Sciences Malaysia.
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Hashim, N.H.F., Mahadi, N.M., Illias, R.M. et al. Biochemical and structural characterization of a novel cold-active esterase-like protein from the psychrophilic yeast Glaciozyma antarctica. Extremophiles 22, 607–616 (2018). https://doi.org/10.1007/s00792-018-1021-z
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DOI: https://doi.org/10.1007/s00792-018-1021-z