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View Preprint Archebacterial Lysyl Oxidase 1 Expression and Properties of Lysyl Oxidase from Archeal Halophile 2 Haloterrigena turkmenica* 3 4 Nikolay B. Pestov1, Daniel V. Kalinovsky1, Tatyana D. Larionova1, Alia Z. Zakirova1, 5 Nikolay N. Modyanov2, Irina A. Okkelman1, Tatyana V. Korneenko1 6 7 1Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia 8 2 University of Toledo College of Medicine, Toledo, Ohio 9 * Running title: Archebacterial Lysyl Oxidase 10 To whom correspondence should be addressed: Shemyakin and Ovchinnikov Institute of 11 Bioorganic Chemistry, Miklukho-Maklaya 16/10, Moscow, 117997, Russia, Tel: +7(495) 330- 12 6556; Fax: +7(495) 330-6556; E-mail: [email protected] 13 Keywords: amine oxidase, horizontal gene transfer, protein cross-linking 14 ABSTRACT 15 Background: Lysyl oxidases (LOX) were studied mostly in mammals, whereas properties of 16 recently found homologs in prokaryotic genomes remain enigmatic. Methods: LOX gene from 17 Haloterrigena turkmenica has been cloned by PCR in a E. coli expression vector. Protein 18 purification has been done using metal affinity chromatography under denaturing conditions 19 followed by refolding. Catalytic activity has been fluorometrically a release of hydrogen 20 peroxide coupled with the oxidation of 10-acetyl-3,7-dihydroxyphenoxazine in the presence of 21 horseradish peroxidase. Rabbit polyclonal antibodies were obtained and used in western blotting. 22 Results: H. turkmenica LOX (HTU-LOX) may be successfully expressed in E. coli with a high 23 yield. However, full-length protein gives no catalytic activity. On the other hand, a deletion of 24 putative signal peptide allows the protein to be refolded into an active enzyme. Further deletion 25 until the boundary of the catalytic C-terminal domain greatly increases the activity. Refolding is 26 optimal at pH around 6.0, with addition of Cu2+, and surprisingly does not respond to changing 27 concentration s of NaCl. The active HTU-LOX is sensitive to the lysyl oxidase inhibitor β- 28 aminopropionitrile. HTU-LOX is active towards usual substrates of mammalian LOX such as 29 lysine-containing basic peptides and polymers. The major difference between HTU-LOX and 30 mammalian LOX is a relaxed specificity of the former. HTU-LOX readily oxidizes various 31 amines including such compounds as taurine and glycine. Moreover, it is active also towards 32 aminoglycoside antibiotics. Benzyl amine is a poor substrate for HTU-LOX. H. turkmenica cells 33 or culture medium do not contain any detectable amine oxidase activity. Polyclonal antibodies 1 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2691v1 | CC BY 4.0 Open Access | rec: 3 Jan 2017, publ: 3 Jan 2017 Archebacterial Lysyl Oxidase 34 against HTU-LOX detect a band among H. turkmenica proteins with the molecular weight 35 corresponding to the unprocessed enzyme. Conclusion: H. turkmenica contains a lysyl oxidase 36 gene that may give active recombinant enzyme with important biochemical features conserved, 37 for example, sensitivity to β-aminopropionitrile. However, its function in the host may be 38 cryptic. Significance: This is the first report on some properties of lysyl oxidase that originated 39 from a horizontal transfer event into Archea. 40 41 42 INTRODUCTION 43 Human genome contains five lysyl oxidase isoforms (LOX and LOXL 1-4), all of them possess 44 the highly conserved C-terminal catalytic domain. It is known that functions of the various 45 isoforms of lysyl oxidase in mammals include remodeling of extracellular matrix (ECM). It can 46 be assumed that LOXL2/3/4 are preferably involved in the formation of cross-linking of collagen 47 basement membranes, whereas isoforms LOX and LOXL15 contributed to maturation of 48 extracellular matrix fibers like elastin and fibronectin in chordates (Grau-Bove et al, 2015). 49 Most other animal genomes contain from one to five lysyl oxidase genes (nematodes being an 50 important exception), whereas plants and most fungi are known to lack it. In the vast majority of 51 prokaryotic genomes lysyl oxidase genes are also absent, then even the more interesting is the 52 presence of true homologues of animal lysyl oxidase in certain species of bacteria. This reflects 53 the unique history of this unique enzyme – it is obvious that lysyl oxidase gene underwent 54 multiple horizontal transfer, and is now common in actinomycetes (especially streptomycetes), 55 many deltaproteobacteria, occasionally – in other eubacteria, and very rarely – among Achaea. 56 This aspect is extremely interesting not only from the phylogenetic point of view, but also 57 because of potential biotechnological applications due to the fact that distantly related enzymes 58 may have peculiarly interesting properties (Grau-Bove et al, 2015). 59 It is interesting to note that in contrast to eukaryotic lysyl oxidase, some lysyl oxidase sequences 60 identified in prokaryotes protein exhibit simple architecture with a single domain exclusively 61 LOX, often do not include a signal peptide (Grau-Bove et al, 2015). On the other hand, some 62 prokaryotic lysyl oxidases are more complex, for example, LOX from Sorangium cellulosum 63 possesses a unique C-terminal non-catalytic domain, putatively highly disulfide cross-linked. 64 The homologues of lysyl oxidase in Archaea are clustered in two independent groups: 65 Thaumarchaeotes and Euryarchaeotes. This suggests that Thaumarchaeotes and Euryarchaeotes 66 may had acquired LOX genes in two independent horizontal transfer (HGT) events (Grau-Bove 67 et al, 2015). 2 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2691v1 | CC BY 4.0 Open Access | rec: 3 Jan 2017, publ: 3 Jan 2017 Archebacterial Lysyl Oxidase 68 Considering specifically the lysyl oxidase from archaeal halophile Haloterrigena turkmenica, it 69 should be emphasized that the complete genome has been sequenced. The archaebacteria 70 genome consists of 5,440,782 base pairs (including plasmid 6). Of these 5,287 encode proteins 71 and RNA encoding 63 (Saunders et al, 2010). Haloterrigena turkmenica was first described by 72 Soviet scientists Zvyagintseva and Tarasov in 1987 and firstly named Halococcus turckmenicus 73 (Zvyagintseva and Tarasov, 1987), the renamed as Haloterrigena (Ventosa et al, 1999). It 74 belongs to the genus of the family Halobacteriaceae typus Euryarchaeota. H. turkmenica was 75 isolated from sulphate saline soil, it is a fairly fast growing, chemoorganotrophic extreme 76 halophile (requires for growth at least 2 M NaCl). 77 Here, we attempted for the first time a study on the properties of lysyl oxidase from this 78 interesting archebacterion. 79 EXPERIMENTAL PROCEDURES 80 Materials and strains. A fresh stock of Haloterrigena turkmenica VKMB-1734 was purchased 81 from All-Russian Collection of Microorganisms (G.K. Skryabin Institute of Biochemistry and 82 Physiology of Microorganisms, Pushchino, Moscow Region, Russia). 83 Cultivation of H. turkmenica. Various haloarcheal media with NaCl around 200 g/l such as INMI 84 medium-3, DSMZ-372 are suitable. Care should be taken to adjust pH since H. turkmenica does 85 not grow in acidic media. We found a simper medium (hereafter referred to as IA) a better 86 choice: casamino acids – 5 g/l, yeast extract – 5 g/l, NaCl – 220 g/l, pH 7.6 - autoclaved and 87 supplemented with MgSO4 – 5 mM, CuCl2 – 10 μM. Also, H. turkmenica can be easily adapted 88 to a defined medium, hereafter referred to as MHTU (Halobacterial minimal medium (HMM) from 89 2014, J. Med. Physiol. Biophys. 6, 42) – modified from L-alanine – 0.4, L-arginine – 0.4, D-asparagine 90 – 0.2, L-aspartic acid – 0.4, L-cysteine – 0.1, L-glutamic acid – 1.5, L-histidine – 0.7, L-isoleucine – 91 0.5, L-leucine – 0.8, D,L-lysine 2, D,L-methionine – 0.4, L-phenylalanine – 0.3, L-proline – 0.4, D,L- 92 serine – 0.6, L-threonine – 1, L-tyrosine – 0.2, D,.L-tryptophan – 0.5, L-valine – 1, AMP – 0.1, NaCl – 93 220, MgSO4-7H2O – 20, KCL – 2, NH4Cl – 0.5, KNO3 – 0.1, KH2PO4 – 0.1, K2HPO4 – 0.1, Na3- 94 citrate – 0.8, MnSO4-2H2O – 0.0003, CaCl2-6H2O – 0.1, ZnSO4-7H2O – 0.00005, FeSO4-7H2O – 95 0.00005, CuCl2 – 10 μM, glycerol – 1, D-leucine-OH – 0.1, norleucine – 0.1, thymine – 0.1, uracil – 96 0.1, pH 7.5 97 Gene cloning. To carry out the polymerase chain reaction with Taq-polymerase, in 25µl total 98 reaction volume the "direct" and "reverse" primers were added to a concentration of 0.8 µM, 10x 99 Taq PCR buffer, 2.5µl 2 mM solution of deoxyribonucleotide triphosphates, 0.1 μl of Taq DNA 3 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2691v1 | CC BY 4.0 Open Access | rec: 3 Jan 2017, publ: 3 Jan 2017 Archebacterial Lysyl Oxidase 100 polymerase, H. turkmenica genomic DNA as a template, and reaction volume was adjusted to the 101 desired water «Milli-Q». For polymerase chain reaction with Phusion-polymerase in a volume of 25 102 μl were mixed the corresponding "direct" and "reverse" primers at a concentration of 0.8 mM, PCR 103 buffer 5x Phusion GC reaction buffer, 2 μl 2,5 mM deoxyribonucleotide solution, 0.2 μl of Phusion 104 DNA polymerase, H. turkmenica genomic DNA as a template. The DNA used as a template for 105 PCR was isolated from the cell culture using a proprietary kit of «ZR Fungal / Bacterial DNA 106 MicroPrep» ( «Zymo Research») according to the manufacturer's instructions. The following 107 cycling parameters were usede: 1. Hot start 95ºC or 98ºC (Taq or Phusion, respectively) 2 min; 2. 108 Denaturation 95ºC or 98ºC (Taq or Phusion, respectively) 30 sec; 3 1 min Annealing at 55ºC 109 55ºC; 4. Elongation 72ºC 2 min. 30 cycles. 5. The final elongation 72ºC 7 min. In both cases 110 amplification was successful giving HTU-AADN and HTU-QVED fragments (800 and 600 bp, 111 respectively).
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