Abstract
Glaciozyma antarctica PI12 is a psychrophilic yeast isolated from Antarctica. In this work, we describe the heterologous production, biochemical properties and in silico structure analysis of an arginase from this yeast (GaArg). GaArg is a metalloenzyme that catalyses the hydrolysis of l-arginine to l-ornithine and urea. The cDNA of GaArg was reversed transcribed, cloned, expressed and purified as a recombinant protein in Escherichia coli. The purified protein was active against l-arginine as its substrate in a reaction at 20 °C, pH 9. At 10–35 °C and pH 7–9, the catalytic activity of the protein was still present around 50%. Mn2+, Ni2+, Co2+ and K+ were able to enhance the enzyme activity more than two-fold, while GaArg is most sensitive to SDS, EDTA and DTT. The predicted structure model of GaArg showed a very similar overall fold with other known arginases. GaArg possesses predominantly smaller and uncharged amino acids, fewer salt bridges, hydrogen bonds and hydrophobic interactions compared to the other counterparts. GaArg is the first reported arginase that is cold-active, facilitated by unique structural characteristics for its adaptation of catalytic functions at low-temperature environments. The structure and function of cold-active GaArg provide insights into the potentiality of new applications in various biotechnology and pharmaceutical industries.
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All sequences and structure coordinates reported are available by their GenBank accession numbers or Protein Data Bank identifier code, unless specifically stated otherwise
References
Alliegro MC (2000) Effects of dithiothreitol on protein activity unrelated to thiol- disulfide exchange: For consideration in the analysis of protein function with cleland’s reagent. Anal Biochem 282(1):102–106. https://doi.org/10.1006/abio.2000.4557
Åqvist J, Kazemi M, Isaksen GV, Brandsdal BO (2017) Entropy and Enzyme Catalysis. Acc Chem Res 50(2):199–207. https://doi.org/10.1021/acs.accounts.6b00321
Ash, DE (2004) Structure and function of arginases j nutr, 134(10 Suppl), 2760S-2764S discussion. 2765S-2767S doi:134/10/2760S
Bewley MC, Lott JS, Baker EN, Patchett ML (1996) The cloning, expression and crystallisation of a thermostable arginase. FEBS Lett 386(2–3):215–218. https://doi.org/10.1016/0014-5793(96)00459-0
Bharudin I, Abu Bakar MF, Hashim NHF, Mat Isa MN, Alias H, Firdaus-Raih M, MdIllias R et al (2018) Unravelling the adaptation strategies employed by Glaciozyma antarctica PI12 on Antarctic sea ice. Mar Environ Res 137:169–176. https://doi.org/10.1016/j.marenvres.2018.03.007
Boo SY, Wong CMVL, Rodrigues KF, Najimudin N, Murad AMA, Mahadi NM (2013) Thermal stress responses in Antarctic yeast, Glaciozyma antarctica PI12, characterized by real-time quantitative PCR. Polar Biol 36(3):381–389. https://doi.org/10.1007/s00300-012-1268-2
Caldwell RW, Rodriguez PC, Toque HA, Priya Narayanan S, Caldwell RB (2018) Arginase: A multifaceted enzyme important in health and disease. Physiol Rev 98(2):641–665. https://doi.org/10.1152/physrev.00037.2016
Carrasco M, Rozas JM, Barahona S, Alcaíno J, Cifuentes V, Baeza M (2012) Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antarctic region. BMC Microbiol 12:251. https://doi.org/10.1186/1471-2180-12-251
Carrillo N, Ceccarelli EA, Roveri OA (2010) Usefulness of kinetic enzyme parameters in biotechnological practice. Biotechnol Genet Eng Rev 27(1):367–382. https://doi.org/10.1080/02648725.2010.10648157
Chiuri R, Maiorano G, Rizzello A, Del Mercato LL, Cingolani R, Rinaldi R, Maffia M et al (2009) Exploring local flexibility/rigidity in psychrophilic and mesophilic carbonic anhydrases. Biophys J 96(4):1586–1596. https://doi.org/10.1016/j.bpj.2008.11.017
Clemente GS, van Waarde A, Antunes IF, Dömling A, Elsinga PH (2020) Arginase as a potential biomarker of disease progression: A molecular imaging perspective. Int J Mol Sci 21(15):1–36. https://doi.org/10.3390/ijms21155291
Di Costanzo L, Sabio G, Mora A, Rodriguez PC, Ochoa AC, Centeno F, Christianson DW (2005) Crystal structure of human arginase I at A resolution and exploration of inhibition in the immune response. Proc Natl Acad Sci USA 102(37):13058–13063. https://doi.org/10.1073/pnas.0504027102
Dabir S, Dabir P, Somvanshi B (2005) Purification, properties and alternate substrate specificities of arginase from two different sources: Vigna catjang cotyledon and buffalo liver. Int J Biol Sci 1(3):114–122. https://doi.org/10.7150/ijbs.1.114
Das P, Lahiri A, Lahiri A, Chakravortty D (2010) Modulation of the arginase pathway in the context of microbial pathogenesis: A metabolic enzyme moonlighting as an immune modulator. PLoS Pathog 6(6):1–7. https://doi.org/10.1371/journal.ppat.1000899
Feller G (2013) Psychrophilic Enzymes: From Folding to Function and Biotechnology. Scientifica 2013:1–28. https://doi.org/10.1155/2013/512840
Fields PA, Somero GN (1998) Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes. Proc Natl Acad Sci USA 95(19):11476–11481. https://doi.org/10.1073/pnas.95.19.11476
Firdaus-Raih M, Hashim NHF, Bharudin I, Bakar MFA, Huang KK, Alias H, Lee BKB et al (2018) The Glaciozyma antarctica genome reveals an array of systems that provide sustained responses towards temperature variations in a persistently cold habitat. PLoS ONE 13(1):1–18. https://doi.org/10.1371/journal.pone.0189947
Fujiu S, Ito M, Kobayashi E, Hanada Y, Yoshida M, Kudoh S, Hoshino T (2021) Basidiomycetous yeast, glaciozyma antarctica, forming frost-columnar colonies on frozen medium. Microorganisms 9(8):1–11. https://doi.org/10.3390/microorganisms9081679
Garcia TA, Ventura CL, Smith MA, Merrell DS, Brien ADO (2013) Cytotoxic Necrotizing Factor 1 and Hemolysin from Uropathogenic Escherichia coli Elicit Different Host Responses in the Murine Bladder. Infect Immun 81(1):99–109. https://doi.org/10.1128/IAI.00605-12
Gaur N, Kumar K, Saxena JK (2019) Molecular characterization of recombinant arginase of Leishmania donovani. Protein Expr Purif 159:1–9. https://doi.org/10.1016/j.pep.2019.02.018
Georlette D, Blaise V, Collins T, D’Amico S, Gratia E, Hoyoux A, Marx JC et al (2004) Some like it cold: Biocatalysis at low temperatures. FEMS Microbiol Rev 28(1):25–42. https://doi.org/10.1016/j.femsre.2003.07.003
Gerday C (2013) Psychrophily and catalysis. Biology 2(2):719–741. https://doi.org/10.3390/biology2020719
Geyer JW, Dabich D (1971) Rapid method for determination of arginase activity in tissue homogenates. Anal Biochem 39(2):412–417. https://doi.org/10.1016/0003-2697(71)90431-3
Gianese G, Argos P, Pascarella S (2001) Structural adaptation of enzymes to low temperatures. Protein Eng 14(3):141–148. https://doi.org/10.1093/protein/14.3.141
Gogoi M, Datey A, Wilson KT, Chakravortty D (2016) Dual Role of Arginine Metabolism in Establishing Pathogenesis. Curr Opin Microbiol 29:43–48. https://doi.org/10.1016/j.mib.2015.10.005
Green SM, Ginsburg A, Lewis MS, Hensley P (1991) Roles of metal ions in the maintenance of the tertiary and quaternary structure of arginase from Saccharomyces cerevisiae. J Biol Chem 266(32):21474–21481. https://doi.org/10.1016/s0021-9258(18)54663-9
Hai Y, Edwards JE, Van Zandt MC, Hoffmann KF, Christianson DW (2014) Crystal structure of Schistosoma mansoni arginase, a potential drug target for the treatment of schistosomiasis. Biochemistry 53(28):4671–4684. https://doi.org/10.1021/bi5004519
Hamid B, Bashir Z, Yatoo AM, Mohiddin F, Majeed N, Bansal M, Poczai P et al (2022) Cold-active enzymes and their potential industrial applications — a review. Molecules 27(18):5885
Hashim NHF, Bharudin I, Nguong DLS, Higa S, Bakar FDA, Nathan S, Rabu A et al (2013) Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12. Extremophiles 17(1):63–73. https://doi.org/10.1007/s00792-012-0494-4
Huang K, Mu W, Zhang T, Jiang B, Miao M (2016a) Cloning, expression, and characterization of a thermostable l-arginase from Geobacillus thermodenitrificans NG80-2 for l-ornithine production. Biotechnol Appl Biochem 63(3):391–397. https://doi.org/10.1002/bab.1385
Huang K, Zhang T, Jiang B, Mu W, Miao M (2016b) Characterization of a thermostable arginase from Rummeliibacillus pycnus SK31.001. J Mol Catal B Enzym 133:S68–S75. https://doi.org/10.1016/j.molcatb.2016.11.020
Ide A, Hernández V, Medina-Aparicio L, Carcamo-Noriega E, Girard L, Hernández-Lucas I, Dunn M (2020) Genetic regulation, biochemical properties and physiological importance of arginase from Sinorhizobium meliloti. Microbiology 166(5):484–497. https://doi.org/10.1099/mic.0.000909
Isaksen GV, Åqvist J, Brandsdal BO (2016) Enzyme surface rigidity tunes the temperature dependence of catalytic rates. Proc Natl Acad Sci USA 113(28):7822–7827. https://doi.org/10.1073/pnas.1605237113
Jaafar NR, Littler D, Beddoe T, Rossjohn J, MdIllias R, Mahadi NM, Mackeen MM et al (2016) Crystal structure of fuculose aldolase from the Antarctic psychrophilic yeast Glaciozyma antarctica PI12. Acta Crystallograph Sect: F Structural Biol Commun 72(11):831–839. https://doi.org/10.1107/S2053230X16015612
Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K et al (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596(7873):583–589. https://doi.org/10.1038/s41586-021-03819-2
Kamaruddin S, Redzuan RA, Minor N, Wan Seman WMK, Md Tab M, Jaafar NR, Rodzli NA et al (2022) Biochemical Characterisation and Structure Determination of a Novel Cold-Active Proline Iminopeptidase from the Psychrophilic Yeast, Glaciozyma antarctica PI12. Catalysts 12(722):1–14
Kövamees O, Shemyakin A, Pernow J (2014) Effect of arginase inhibition on ischemia-reperfusion injury in patients with coronary artery disease with and without diabetes mellitus. PLoS ONE 9(7):1–7. https://doi.org/10.1371/journal.pone.0103260
Li Z, Wang L, Ren Y, Huang Y, Liu W, Lv Z, Qian L et al (2022) Arginase: shedding light on the mechanisms and opportunities in cardiovascular diseases. Cell Death Discover 8(1):1–14. https://doi.org/10.1038/s41420-022-01200-4
Lonhienne T, Gerday C, Feller G (2000) Psychrophilic enzymes: Revisiting the thermodynamic parameters of activation may explain local flexibility. Biochim Et Biophys Acta - Protein Struct and Mol Enzymol 1543(1):1–10. https://doi.org/10.1016/S0167-4838(00)00210-7
López V, Alarcón R, Orellana MS, Enríquez P, Uribe E, Martínez J, Carvajal N (2005) Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies: Alteration of substrate specificity by replacement of Asn149 with Asp. FEBS J 272(17):4540–4548. https://doi.org/10.1111/j.1742-4658.2005.04874.x
McGee DJ, Radcliff FJ, Mendz GL, Ferrero RL, Mobley HLT (1999) Helicobacter pylori rocF is required for arginase activity and acid protection in vitro but is not essential for colonization of mice or for urease activity. J Bacteriol 181(23):7314–7322
McGee DJ, Zabaleta J, Viator RJ, Testerman TL, Ochoa AC, Mendz GL (2004) Purification and characterization of Helicobacter pylori arginase, RocF: Unique features among the arginase superfamily. Eur J Biochem 271(10):1952–1962. https://doi.org/10.1111/j.1432-1033.2004.04105.x
McNicholas S, Potterton E, Wilson KS, Noble MEM (2011) Presenting your structures: the CCP4mg molecular-graphics software. Acta Crystallogr D Biol Crystallogr. https://doi.org/10.1107/S0907444911007281
Mendz GL, Holmes EM, Ferrero RL (1998) In situ characterization of Helicobacter pylori arginase. Biochim Et Biophys Acta - Protein Struct and Mol Enzymol 1388(2):465–477. https://doi.org/10.1016/S0167-4838(98)00207-6
Mohamed SA, Fahmy AS, Mohamed TM, Hamdy SM (2005) Urea cycle of Fasciola gigantica: Purification and characterization of arginase. Comparative Biochem and Physiol - B Biochem and Mol Biol 142(3):308–316. https://doi.org/10.1016/j.cbpb.2005.08.002
Moreno-Vivián C, Soler G, Castillo F (1992) Arginine catabolism in the phototrophic bacterium Rhodobacter capsulatus E1F1: Purification and properties of arginase. Eur J Biochem 204(2):531–537. https://doi.org/10.1111/j.1432-1033.1992.tb16664.x
Muhamad Ismail NAS, Yap SH, Mohamad Yussoff MA, Nor Muhammad NA, Firdaus-Raih M, Quay DHX (2022) Modeling and computational characterization of a Xanthomonas sp Hypothetical protein identifies a remote ortholog of Burkholderia lethal factor 1. J Biomol Struct Dyn. https://doi.org/10.1080/07391102.2022.2100827
Niu F, Yu Y, Li Z, Ren Y, Li Z, Ye Q, Liu P et al (2022) Arginase: An emerging and promising therapeutic target for cancer treatment. Biomed Pharmacother. https://doi.org/10.1016/j.biopha.2022.112840
Palamini M, Canciani A, Forneris F (2016) Identifying and visualizing macromolecular flexibility in structural biology. Front Mol Biosci. https://doi.org/10.3389/fmolb.2016.00047
Parvizpour S, Hussin N, Shamsir MS, Razmara J (2021) Psychrophilic enzymes: structural adaptation, pharmaceutical and industrial applications. Appl Microbiol Biotechnol 105(3):899–907. https://doi.org/10.1007/s00253-020-11074-0
Patchett ML, Daniel RM, Morgan HW (1991) Characterisation of arginase from the extreme thermophile “Bacillus caldovelox.” Biochim Et Biophys Acta (BBA)/protein Struct and Mol 1077(3):291–298. https://doi.org/10.1016/0167-4838(91)90543-9
Permyakov EA (2021) Metal binding proteins. Encyclopedia 1(1):261–292. https://doi.org/10.3390/encyclopedia1010024
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF chimera - a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612. https://doi.org/10.1002/jcc.20084
Prejanò M, Alberto ME, Russo N, Toscano M, Marino T (2020) The effects of the metal ion substitution into the active site of metalloenzymes: a theoretical insight on some selected cases. Catalysts 10(9):1–28. https://doi.org/10.3390/catal10091038
Reczkowski RS, Ash DE (1994) Rat liver arginase: kinetic mechanism, alternate substrates, and inhibitors. Arch Biochem Biophys. https://doi.org/10.1006/abbi.1994.1276
Riess C, Shokraie F, Classen CF, Kreikemeyer B, Fiedler T, Junghanss C, Maletzki C (2018) Arginine-depleting enzymes - an increasingly recognized treatment strategy for therapy-refractory malignancies. Cell Physiol Biochem 51(2):854–870. https://doi.org/10.1159/000495382
Riley E, Roberts SC, Ullman B (2011) Inhibition profile of Leishmania mexicana arginase reveals differences with human arginase I. Int J Parasitol 41(5):545–552. https://doi.org/10.1016/j.ijpara.2010.12.006
Ruff KM, Pappu RV (2021) AlphaFold and Implications for intrinsically disordered proteins. J Mol Biol 433(20):167208. https://doi.org/10.1016/j.jmb.2021.167208
Sadarangani V, Rahman S, Sau AK (2018) Metal ions-induced stability and function of bimetallic human arginase-I, a therapeutically important enzyme. Biochim Et Biophys Acta - Proteins and Proteom 1866(11):1153–1164. https://doi.org/10.1016/j.bbapap.2018.08.006
Shimotohno K, Iida J, Takizawa N, Endō T (1994) Purification and Characterization of Arginine Amidinohydrolase from Bacillus brevis TT02-8. Biosci Biotechnol Biochem 58(6):1045–1049. https://doi.org/10.1271/bbb.58.1045
Sočan J, Isaksen GV, Brandsdal BO, Åqvist J (2019) Towards Rational Computational Engineering of Psychrophilic Enzymes. Sci Rep 9(1):1–10. https://doi.org/10.1038/s41598-019-55697-4
Stark C, Bautista-Leung T, Siegfried J, Herschlag D (2022) System investigation of the link between enzyme catalysis and cold adaptation. eLife 11(1):1–14. https://doi.org/10.7554/eLife.72884
Struvay C, Feller G (2012) Optimization to low temperature activity in psychrophilic enzymes. Int J Mol Sci 13(9):11643–11665. https://doi.org/10.3390/ijms130911643
Turchetti B, Hall SRT, Connell LB, Branda E, Buzzini P, Theelen B, Müller WH et al (2011) Psychrophilic yeasts from Antarctica and European glaciers: Description of Glaciozyma gen. nov., Glaciozyma martinii sp. nov. and Glaciozyma watsonii sp nov. Extremophiles 15(5):573–586. https://doi.org/10.1007/s00792-011-0388-x
Uthurry CA, Suárez Lepe JA, Lombardero J, García Del Hierro JR (2006) Ethyl carbamate production by selected yeasts and lactic acid bacteria in red wine. Food Chem 94(2):262–270. https://doi.org/10.1016/j.foodchem.2004.11.017
van Kempen M, Kim SS, Tumescheit C, Mirdita M, Gilchrist CLM, Söding J, Steinegger M (2022) Foldseek: fast and accurate protein structure search. BioRxiv. https://doi.org/10.1038/s41587-023-01773-0
Viator RJ, Rest RF, Hildebrandt E, McGee DJ (2008) Characterization of Bacillus anthracis arginase: Effects of pH, temperature, and cell viability on metal preference. BMC Biochem 9(1):1–14. https://doi.org/10.1186/1471-2091-9-15
Yang Z, Huang Z, Wu Q, Tang X, Huang Z (2023) Cold-adapted proteases : an efficient and energy-saving biocatalyst. Int J Mol Sci 24(10):8532
Yu JJ, Park KB, Kim SG, Oh SH (2013) Expression, purification, and biochemical properties of arginase from bacillus subtilis 168. J Microbiol 51(2):222–228. https://doi.org/10.1007/s12275-013-2669-9
Yusof NA, Hashim NHF, Bharudin I (2021) Cold adaptation strategies and the potential of psychrophilic enzymes from the antarctic yeast, glaciozyma antarctica pi12. J of Fungi. https://doi.org/10.3390/jof7070528
Zeng R, Zhang R, Zhao J, Lin N (2003) Cold-active serine alkaline protease from the psychrophilic bacterium Pseudomonas strain DY-A: Enzyme purification and characterization. Extremophiles 7(4):335–337. https://doi.org/10.1007/s00792-003-0323-x
Zhang J, Zhang X, Wu C, Lu D, Guo G, Mao X, Zhang Y et al (2011) Expression, purification and characterization of arginase from helicobacter pylori in its apo form. PLoS ONE 6(10):1–8. https://doi.org/10.1371/journal.pone.0026205
Zhang T, Guo Y, Zhang H, Mu W, Miao M, Jiang B (2013) Arginase from Bacillus thuringiensis SK 20.001: Purification, characteristics, and implications for l-ornithine biosynthesis. Process Biochem 48(4):663–668. https://doi.org/10.1016/j.procbio.2013.02.023
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We are grateful to the Malaysia Genome and Vaccine Institute (MGVI) for the use of facilities and staff support. This work was supported by the Ministry of Science, Technology and Innovation of Malaysia (MOSTI) [grant number 02-05-20-SF007/2].
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Ministry of Science, Technology and Innovation of Malaysia (MOSTI), grant number 02-05-20-SF007/2
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Conceptualization: AMAM: methodology: NYY, DQHX: formal analysis and investigation: NYY, DQHX: writing—original draft preparation: NYY, DQHX: writing—review and editing: DQHX, NYY: funding acquisition: AMAM: resources: AMAM, MAJ: supervision: AMAM, FDAB, SK, RMIl, MFR, NMM.
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Yusof, N.Y., Quay, D.H.X., Kamaruddin, S. et al. Biochemical and in silico structural characterization of a cold-active arginase from the psychrophilic yeast, Glaciozyma antarctica PI12. Extremophiles 28, 15 (2024). https://doi.org/10.1007/s00792-024-01333-7
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DOI: https://doi.org/10.1007/s00792-024-01333-7