Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1
Abstract
:1. Introduction
2. Results
2.1. The Temporal Stability of the crARC Activity
2.2. The Interaction of crARC, crCytb5-1 and crCytb5-R
2.3. Study of Different crARC Variants
2.4. The Reduction Activity of the crARC Variants
2.5. The Mo Cofactor Content of the crARC Variants
2.6. The Oligomerization of the crARC Variants
3. Discussion
4. Materials and Methods
4.1. Chemicals
4.2. Bacterial Strains and Culture Conditions
4.3. Cloning of cDNA for Recombinant Protein Expression.
4.4. Expression and Purification of Recombinant Proteins
4.5. DNA Sequencing and Sequence Analysis
4.6. HAP and Adenine Quantification
4.7. Benzamidine and Benzamidoxime Quantification
4.8. The Quantification of the crARC Reduction Activity
4.9. Determination of the Organic Motive of Mo Cofactor
4.10. Molecular Weight Determination by SEC
4.11. Studies of Tertiary and Quaternary Structure of crARC Mutants
4.12. Software Used to Predict the Three-Dimensional Structure of crARC
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
- Schwarz, G.; Mendel, R.R.; Ribbe, M.W. Molybdenum cofactors, enzymes and pathways. Nature 2009, 460, 839–847. [Google Scholar] [CrossRef] [PubMed]
- Mendel, R.R.; Leimkuhler, S. The biosynthesis of the molybdenum cofactors. J. Biol. Inorg. Chem. 2015, 20, 337–347. [Google Scholar] [CrossRef] [PubMed]
- Llamas, A.; Tejada-Jimenez, M.; Fernandez, E.; Galvan, A. Molybdenum metabolism in the alga Chlamydomonas stands at the crossroad of those in Arabidopsis and humans. Metallomics 2011, 3, 578–590. [Google Scholar] [CrossRef] [PubMed]
- Havemeyer, A.; Bittner, F.; Wollers, S.; Mendel, R.; Kunze, T.; Clement, B. Identification of the missing component in the mitochondrial benzamidoxime prodrug-converting system as a novel molybdenum enzyme. J. Biol. Chem. 2006, 281, 34796–34802. [Google Scholar] [CrossRef] [PubMed]
- Anantharaman, V.; Aravind, L. Mosc domains: Ancient, predicted sulfur-carrier domains, present in diverse metal-sulfur cluster biosynthesis proteins including molybdenum cofactor sulfurases. FEMS Microbiol. Lett. 2002, 207, 55–61. [Google Scholar] [CrossRef]
- Kozmin, S.G.; Leroy, P.; Pavlov, Y.I.; Schaaper, R.M. YcbX and Yiim, two novel determinants for resistance of Escherichia coli to N-hydroxylated base analogues. Mol. Microbiol. 2008, 68, 51–65. [Google Scholar] [CrossRef] [PubMed]
- Ott, G.; Havemeyer, A.; Clement, B. The mammalian molybdenum enzymes of mARC. J. Biol. Inorg. Chem. 2015, 20, 265–275. [Google Scholar] [CrossRef] [PubMed]
- Arc, E.; Galland, M.; Godin, B.; Cueff, G.; Rajjou, L. Nitric oxide implication in the control of seed dormancy and germination. Front. Plant Sci. 2013, 4. [Google Scholar] [CrossRef] [PubMed]
- Wahl, B.; Reichmann, D.; Niks, D.; Krompholz, N.; Havemeyer, A.; Clement, B.; Messerschmidt, T.; Rothkegel, M.; Biester, H.; Hille, R.; et al. Biochemical and spectroscopic characterization of the human mitochondrial amidoxime reducing components hmARC-1 and hmARC-2 suggests the existence of a new molybdenum enzyme family in eukaryotes. J. Biol. Chem. 2010, 285, 37847–37859. [Google Scholar] [CrossRef] [PubMed]
- Krompholz, N.; Krischkowski, C.; Reichmann, D.; Garbe-Schönberg, D.; Mendel, R.R.; Bittner, F.; Clement, B.; Havemeyer, A. The mitochondrial amidoxime reducing component (mARC) is involved in detoxification of N-hydroxylated base analogues. Chem. Res. Toxicol. 2012, 25, 2443–2450. [Google Scholar] [CrossRef] [PubMed]
- Von Wirén, N.; Merrick, M. Regulation and function of ammonium carriers in bacteria, fungi, and plants. In Molecular Mechanisms Controlling Transmembrane Transport; Springer: Berlin, Germany, 2004; pp. 95–120. [Google Scholar]
- Kozmin, S.G.; Schaaper, R.M. Molybdenum cofactor-dependent resistance to N-hydroxylated base analogs in Escherichia coli is independent of MobA function. Mutat. Res. 2007, 619, 9–15. [Google Scholar] [CrossRef] [PubMed]
- Tejada-Jimenez, M.; Chamizo-Ampudia, A.; Galvan, A.; Fernandez, E.; Llamas, A. Molybdenum metabolism in plants. Metallomics 2013, 5, 1191–1203. [Google Scholar] [CrossRef] [PubMed]
- Plitzko, B.; Ott, G.; Reichmann, D.; Henderson, C.J.; Wolf, C.R.; Mendel, R.; Bittner, F.; Clement, B.; Havemeyer, A. The involvement of mitochondrial amidoxime reducing components 1 and 2 and mitochondrial cytochrome b5 in N-reductive metabolism in human cells. J. Biol. Chem. 2013, 288, 20228–20237. [Google Scholar] [CrossRef] [PubMed]
- Plitzko, B.; Havemeyer, A.; Bork, B.; Bittner, F.; Mendel, R.; Clement, B. Defining the role of the NADH-cytochrome-b5 reductase 3 in the mitochondrial amidoxime reducing component enzyme system. Drug Metab. Dispos. 2016, 44, 1617–1621. [Google Scholar] [CrossRef] [PubMed]
- Kozmin, S.G.; Wang, J.; Schaaper, R.M. Role for CysJ flavin reductase in molybdenum cofactor-dependent resistance of Escherichia coli to 6-N-hydroxylaminopurine. J. Bacteriol. 2010, 192, 2026–2033. [Google Scholar] [CrossRef] [PubMed]
- Chamizo-Ampudia, A.; Galvan, A.; Fernandez, E.; Llamas, A. The chlamydomonas reinhardtii molybdenum cofactor enzyme crARC has a Zn-dependent activity and protein partners similar to those of its human homologue. Eukaryotic Cell 2011, 10, 1270–1282. [Google Scholar] [CrossRef] [PubMed]
- Sparacino-Watkins, C.E.; Tejero, J.; Sun, B.; Gauthier, M.C.; Thomas, J.; Ragireddy, V.; Merchant, B.A.; Wang, J.; Azarov, I.; Basu, P.; et al. Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2. J. Biol. Chem. 2014, 289, 10345–10358. [Google Scholar] [CrossRef] [PubMed]
- Chamizo-Ampudia, A.; Sanz-Luque, E.; Llamas, Á.; Ocaña-Calahorro, F.; Mariscal, V.; Carreras, A.; Barroso, J.B.; Galván, A.; Fernández, E. A dual system formed by the ARC and NR molybdoenzymes mediates nitrite-dependent NO production in Chlamydomonas. Plant Cell Environ. 2016, 39, 2097–2107. [Google Scholar] [CrossRef] [PubMed]
- Kotthaus, J.; Wahl, B.; Havemeyer, A.; Schade, D.; Garbe-Schönberg, D.; Mendel, R.; Bittner, F.; Clement, B. Reduction of Nω-hydroxy-l-arginine by the mitochondrial amidoxime reducing component (mARC). Biochem. J. 2011, 433, 383–391. [Google Scholar] [CrossRef] [PubMed]
- Mur, L.A.J.; Mandon, J.; Persijn, S.; Cristescu, S.M.; Moshkov, I.E.; Novikova, G.V.; Hall, M.A.; Harren, F.J.M.; Hebelstrup, K.H.; Gupta, K.J. Nitric oxide in plants: An assessment of the current state of knowledge. Aob. Plants 2013, 5. [Google Scholar] [CrossRef] [PubMed]
- Ataya, F.S.; Witte, C.P.; Galván, A.; Igeño, M.I.; Fernández, E. Mcp1 encodes the molybdenum cofactor carrier protein in Chlamydomonas reinhardtii and participates in protection, binding, and storage functions of the cofactor. J. Biol. Chem. 2003, 278, 10885–10890. [Google Scholar] [CrossRef] [PubMed]
- Fischer, K.; Llamas, A.; Tejada-Jimenez, M.; Schrader, N.; Kuper, J.; Ataya, F.S.; Galvan, A.; Mendel, R.R.; Fernandez, E.; Schwarz, G. Function and structure of the molybdenum cofactor carrier protein from Chlamydomonas reinhardtii. J. Biol. Chem. 2006, 281, 30186–30194. [Google Scholar] [CrossRef] [PubMed]
- Andrews, P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. J. 1964, 91, 222–233. [Google Scholar] [CrossRef] [PubMed]
- Hong, P.; Koza, S.; Bouvier, E.S. Size-exclusion chromatography for the analysis of protein biotherapeutics and their aggregates. J. Liq. Chromatogr. Relat. Technol. 2012, 35, 2923–2950. [Google Scholar] [PubMed]
- Gruenewald, S.; Wahl, B.; Bittner, F.; Hungeling, H.; Kanzow, S.; Kotthaus, J.; Schwering, U.; Mendel, R.R.; Clement, B. The fourth molybdenum containing enzyme mARC: Cloning and involvement in the activation of N-hydroxylated prodrugs. J. Med. Chem. 2008, 51, 8173–8177. [Google Scholar] [CrossRef] [PubMed]
- Klein, J.M.; Busch, J.D.; Potting, C.; Baker, M.J.; Langer, T.; Schwarz, G. The mitochondrial amidoxime-reducing component (mARC1) is a novel signal-anchored protein of the outer mitochondrial membrane. J. Biol. Chem. 2012, 287, 42795–42803. [Google Scholar] [CrossRef] [PubMed]
- Wahl, R.C.; Hageman, R.V.; Rajagopalan, K.V. The relationship of Mo, molybdopterin, and the cyanolyzable sulfur in the Mo cofactor. Arch. Biochem. Biophys. 1984, 230, 264–273. [Google Scholar] [CrossRef]
- Hawkes, T.R.; Bray, R.C. Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase. Biochem. J. 1984, 219, 481–493. [Google Scholar] [CrossRef] [PubMed]
- Campbell, W.H. Structure and function of eukaryotic NAD(P)H: Nitrate reductase. Cell. Mol. Life Sci. 2001, 58, 194–204. [Google Scholar] [CrossRef] [PubMed]
- Neve, E.P.; Nordling, A.; Andersson, T.B.; Hellman, U.; Diczfalusy, U.; Johansson, I.; Ingelman-Sundberg, M. Amidoxime reductase system containing cytochrome b5 type B (CYB5B) and MOSC2 is of importance for lipid synthesis in adipocyte mitochondria. J. Biol. Chem. 2012, 287, 6307–6317. [Google Scholar] [CrossRef] [PubMed]
- Da Cruz, S.; Xenarios, I.; Langridge, J.; Vilbois, F.; Parone, P.A.; Martinou, J.C. Proteomic analysis of the mouse liver mitochondrial inner membrane. J. Biol. Chem. 2003, 278, 41566–41571. [Google Scholar] [CrossRef] [PubMed]
- Islinger, M.; Lüers, G.H.; Li, K.W.; Loos, M.; Völkl, A. Rat liver peroxisomes after fibrate treatment. A survey using quantitative mass spectrometry. J. Biol. Chem. 2007, 282, 23055–23069. [Google Scholar] [CrossRef] [PubMed]
- D’Arrigo, A.; Manera, E.; Longhi, R.; Borgese, N. The specific subcellular localization of two isoforms of cytochrome b5 suggests novel targeting pathways. J. Biol. Chem. 1993, 268, 2802–2808. [Google Scholar] [PubMed]
- Sanz-Luque, E.; Ocana-Calahorro, F.; de Montaigu, A.; Chamizo-Ampudia, A.; Llamas, A.; Galvan, A.; Fernandez, E. THB1, a truncated hemoglobin, modulates nitric oxide levels and nitrate reductase activity. Plant J. 2015, 81, 467–479. [Google Scholar] [CrossRef] [PubMed]
- Belaidi, A.A.; Röper, J.; Arjune, S.; Krizowski, S.; Trifunovic, A.; Schwarz, G. Oxygen reactivity of mammalian sulfite oxidase provides a concept for the treatment of sulfite oxidase deficiency. Biochem. J. 2015, 469, 211–221. [Google Scholar] [CrossRef] [PubMed]
- Ott, G.; Plitzko, B.; Krischkowski, C.; Reichmann, D.; Bittner, F.; Mendel, R.R.; Kunze, T.; Clement, B.; Havemeyer, A. Reduction of sulfamethoxazole hydroxylamine (SMX-HA) by the mitochondrial amidoxime reducing component (mARC). Chem. Res. Toxicol. 2014, 27, 1687–1695. [Google Scholar] [CrossRef] [PubMed]
- Jakobs, H.H.; Mikula, M.; Havemeyer, A.; Strzalkowska, A.; Borowa-Chmielak, M.; Dzwonek, A.; Gajewska, M.; Hennig, E.E.; Ostrowski, J.; Clement, B. The N-reductive system composed of mitochondrial amidoxime reducing component (mARC), cytochrome b5 (CYB5B) and cytochrome b5 reductase (CYB5R) is regulated by fasting and high fat diet in mice. PLoS ONE 2014, 9. [Google Scholar] [CrossRef] [PubMed]
- Malik, A.N.; Rossios, C.; Al-Kafaji, G.; Shah, A.; Page, R.A. Glucose regulation of CDK7, a putative thiol related gene, in experimental diabetic nephropathy. Biochem. Biophys. Res. Commun. 2007, 357, 237–244. [Google Scholar] [CrossRef] [PubMed]
- Temple, C.A.; Graf, T.N.; Rajagopalan, K.V. Optimization of expression of human sulfite oxidase and its molybdenum domain. Arch. Biochem. Biophys. 2000, 383, 281–287. [Google Scholar] [CrossRef] [PubMed]
- Grodberg, J.; Dunn, J.J. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J. Bacteriol. 1988, 170, 1245–1253. [Google Scholar] [CrossRef] [PubMed]
- Zheng, L.; Baumann, U.; Reymond, J.L. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 2004, 32. [Google Scholar] [CrossRef] [PubMed]
- Pace, C.N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995, 4, 2411–2423. [Google Scholar] [CrossRef] [PubMed]
- Llamas, A.; Mendel, R.; Schwarz, N. Synthesis of adenylated molybdopterin—An essential step for molybdenum insertion. J. Biol. Chem. 2004, 279, 55241–55246. [Google Scholar] [CrossRef] [PubMed]
- Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef]
- Eftink, M.R. Fluorescence techniques for studying protein structure. Methods Biochem. Anal. 1991, 35, 127–205. [Google Scholar] [PubMed]
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Chamizo-Ampudia, A.; Galvan, A.; Fernandez, E.; Llamas, A. Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1. Int. J. Mol. Sci. 2017, 18, 670. https://doi.org/10.3390/ijms18030670
Chamizo-Ampudia A, Galvan A, Fernandez E, Llamas A. Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1. International Journal of Molecular Sciences. 2017; 18(3):670. https://doi.org/10.3390/ijms18030670
Chicago/Turabian StyleChamizo-Ampudia, Alejandro, Aurora Galvan, Emilio Fernandez, and Angel Llamas. 2017. "Study of Different Variants of Mo Enzyme crARC and the Interaction with Its Partners crCytb5-R and crCytb5-1" International Journal of Molecular Sciences 18, no. 3: 670. https://doi.org/10.3390/ijms18030670