Archebacterial Lysyl Oxidase

1 Expression and Properties of Lysyl Oxidase from Archeal

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 . 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 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).

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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 turckmenicus 73 (Zvyagintseva and Tarasov, 1987), the renamed as Haloterrigena (Ventosa et al, 1999). It 74 belongs to the genus of the family typus . 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

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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). For further work was done using Pfu-polymerase, because of its smaller tendency to 112 errors.Purified polynucleotide fragments HTU-AADN and HTU-QVED were digested with BamH 113 I and Hind III restriction enzymes and ligated into the vector pQE-30 (BamH I and Hind III sites) 114 and then subjected to transformation of E. coli strain XL1-Blue by electroporation. Colonies 115 screening was performed by PCR . Sequencing was performed using Sanger method.

116 Protein expression. Cell cultures of E. coli XL-1 Blue containing the vectors were incubated on a 117 shaker at 37ºC in LB-medium with ampicillin until OD = 0.7, and expression induced with IPTG 118 for 3 hours after which the cells were centrifuged and stored in a freezer. lysed. Proteins were 119 purified under denaturing conditions (8 M urea) using metal-chelating sorbent Ni-NTA agarose. 120 [30]. The resulting proteins in 8M urea pH 6.3 buffered with 0.5 M imidazole, 0.1 M sodium 121 phosphate and 20 mM tris were dialyzed against corresponding buffers as described in Results.

122 Activity assays.

123 Determination of substrate specificity were performed using a fluorometric method suitable for 124 various amine oxidases as the release of hydrogen peroxide coupled to the oxidation of 10-acetyl- 125 3,7-dihydroxyphenoxazine (reagent Amplex Red) in the presence of horseradish peroxidase. 126 Resulting reaction product (resorufin) has a maximum absorption intensity and emission, 127 respectively, at 571 and 585 nm and was assayed with a Microplate analyzer "Fusion" (Perkin 128 Elmer, USA) at excitation and emission west to 535 and 620 nm, respectively. More specifically, 129 the reaction was carried out in 0.1 M borate buffer pH 8.3 in the presence of 1 U / ml horseradish 130 peroxidase. As a negative control, β-aminopropionitrile was used, a specific inhibitor of lysyl 131 oxidase activity

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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 132 Immunization of rabbits was carried out with purified folded proteins HTU-QVED. The first 133 injection was made with Freund's complete adjuvant, which increases the immunogenicity of the 134 injected emulsion. The second immunization was done with incomplete Freund's adjuvant, 5 weeks 135 after the first. The third, booster infection was done after 6 weeks without adjuvants - around 250 136 microgram antigen was administered. After 1 week from the third immunization, sera were 137 collected and stored with sodium azide as a preservative at 4ºC. Immunization of rabbits has been 138 approved by Animal Care and Use Review Board of Shemyakin-Ovchinnikov Institute of 139 Bioorganic Chemisrty, protocol No 15/2011.

140 Western blotting. After electrophoresis on SDS PAGE Laemmli gels, samples of the H. turkmenica 141 proteins and colored markers length proteins (Amersham Full-Range Rainbow Molecular Weight 142 Markers, «GE Healthcare») were transferred from polyacrylamide gel onto a PVDF membrane. 143 After transfer, the membrane was incubated in buffer TBST with 4% skimmed milk powder, 0.02% 144 NaN3 overnight at 4ºC with shaking in order to suppress nonspecific adsorption. The membrane 145 was incubated with mild agitation on a shaker at room temperature for one hour in 10 ml of TBST 146 buffer solution with primary rabbit antibodies (1: 6000). The membrane was then washed 4 times 147 for 5 minutes, incubating with shaking at room temperature in TBST, and placed in a solution of 10 148 ml of TBST supplemented with secondary antibodies (1: 30,000), which was stirred on a shaker at 149 room temperature for 30 min. Then the membrane was again washed with TBST four times for 5 150 min. Chemiluminescence was recorded using Amersham ECL Western Blotting Detection Reagent, 151 «GE Healthcare.

152 RESULTS AND DISCUSSION

153 Three HTU-LOX proteins have been expressed. The full-length protein produces inclusion 154 bodies without any catalytic activity. For this reason, we proceeded to deletion mutants with 155 subsequent purification under denaturing conditions and refolding. The purity of the resulting 156 eluate was checked by SDS PAGE. Figure 1 represents a gel with target proteins HTU-QVED 157 and HTU-AADN (24.3 and 30.2 kDa, respectively). Since the isoelectric points of the test

158 polypeptides are pI1 = 4.19 and pI2 = 4.58, the observed values of their molecular weights 159 deviate from theoretical values of 46 and 34 kDa, respectively. It was concluded that the derived 160 target recombinant proteins are of sufficient purity.

161 Refolding of the proteins HTU-AA and HTU-QV was done using dialysis against different 162 buffer systems. We investigated a variety of factors that may affect the formation of proteins 163 HTU-AADN and HTU-QVED in native conformation. We studied the effect of the following 164 factors: the type and concentration of buffer (Tris, PBS, acetate), the ionic strength of the 5

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 165 solution (NaCl), the temperature, the effect of metals (Cu, Fe, Zn, Ni, Co, Mn) in different 166 concentrations, pH of the solution (5.0-8.0), dialysis, with a gradual decrease in the 167 concentration of denaturing factor (urea). Optimal pH is around 6.0 (Table 1).

168 It is known that human LOX require the presence of copper ion in the catalytic domain to exhibit 169 amine oxidase activity. For HTU-LOX, indeed, only Cu2+ increases activity, whereas a mixture 170 of different ions paradoxically results in a decrease (Tables 2 and 3).

pH 7.0 6.5 6.0 5.5 5.0 - BAPN 4471 14534 23241 19079 3533 + BAPN 2112 2620 2602 2575 2153 171 Table 1. pH dependence of catalytic activity of HTU-LOX, variant 172 HTU-QVED (artbitrary units).

Ion: – Cu2+ Zn2+ Fe2+ Ni2+ Co2+ Mn2+ all

- BAPN 6236 13513 6408 5309 6961 6705 6498 3311

+ BAPN 2662 3103 2845 2794 2825 2968 2908 3325

173 Table 2. Effect of different ions (1 mM) on refolding of HTU-LOX, variant 174 HTU-QVED, amine oxidase activity of (artbitrary units).

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C (Cu2+) 0 0.1 мкМ 1 мкМ 10 мкМ - BAPN 18288 28023 35576 30847 + BAPN 3144 3621 4041 2345 176 Table 3. Effect of different concentrations of Cu2+ on refolding of HTU- 177 LOX, variant HTU-QVED, amine oxidase activity of (artbitrary units).

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С (NaCl) 0 25 мМ 100 мМ 400 мМ - BAPN 22902 19221 17558 25322 + BAPN 2216 2030 1872 2094 179 Table 4. Effect of NaCl on refolding of HTU-LOX, variant 180 HTU-QVED, amine oxidase activity of (artbitrary units).

181 It is interesting to note that although Haloterrigena turkmenica is an extreme halophile that 182 requires for growth at least 2 M NaCl, refolding is not dependent on the concentration of NaCl 183 (Table 4).

184 As a result, the optimal folding conditions were established. Under these conditions amine 185 oxidase activity towards histamine in protein HTU-QVED is 15 times higher than HTU-AADN. 6

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 186 Optimal conditions folding HTU-AADN and HTU-QVED:

187 • Dialysis against 40 mM acetate buffer, pH 6.0, 1 mM Cu2+ over 3 hours, t = 4ºC;

188 • Dialysis against 40 mM acetate buffer, pH 6.0 for 12 hours, t = 4ºC.

189 The findings can be considered a success, because such values a were never been observed for a 190 recombinant prokaryotic lysyl oxidase.

191 Refolded proteins HTU-QVED and HTU-AADN exhibit activity against a wide variety of 192 primary amines: histamine, methylamine, agmatine, D,L-lysine, cadaverine, ethanolamine, 193 tyramine. Even glycine, β-alanine and taurine are efficiently oxidized, in contrast with 194 mammalian LOX. Our enzymes able to oxidize glycine, β-alanine, γ-aminobutyric acid, and 195 taurine generally is one of the best substrates, and it is very unusual, because the presence of any 196 acidic groups in the vicinity of the amino group is almost completely prevents the oxidation of 197 other Cu-dependent amine oxidase. Further, our enzymes oxidize aminoglycoside antibiotics 198 such as capreomycin! With regard to various proteins, lysine-containing peptides and polymers 199 (e.g., poly-L-lysine), the HTU-LOX behaves almost like enzyme from the aorta. Thus, it is safe 200 to conclude that the LOX HTU has the relaxed substrate specificity. The only amine, which was 201 oxidized worse by HTU-LOX, other than Cu-dependent amine oxidase is benzylamine. Further, 202 the HTU-LOX demonstrated sensitivity to an inhibitor of LOX human β-aminopropionitrile 203 (BAPN).

204 At different stages of crop growth, aliquots were taken and prepared samples of cell lysate and 205 supernatant for protein immunoblotting. Noncompetitive western blot (Figure 2) clearly showed 206 the presence of lysyl oxidase in cell lysate, but none in the culture medium. Full-size H. 207 turkmenica lysyl oxidase theoretically contains 308 amino acids with a molecular weight of 208 33829 Da. the detected protein band has a complexes have large molecular weights and 209 interaktoma reflect the specifics of a given protein. Therefore, it can be concluded that the 210 archaea H. turkmenica clearly expresses its lysyl oxidase.

211 Also, with the help of a protein immunoblotting it was analyzed for the presence of cell lysate 212 and the intensity of expression of lysyl oxidase on different growth stages. There is no 213 relationship between growth stage and the intensity of expression of LOX. Other factors were 214 investigated, which could potentially affect the expression of lysyl oxidase in H. turkmenica. In 215 particular, it has been shown that expression is higher when copper in low concentrations was 216 added to the culture of H. turkmenica. Taking into account the abnormal mobility of the acidic 217 protein it is possible to say that this band corresponds to the expected, but only under the 7

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 218 assumption that there is no processing. Moreover, there is also a positive antibody response to 219 proteins with very low mobility in the area of molecular weights of about 200 kDa. This fact has 220 not yet found an explanation. In the culture medium LOX protein was not detected, i.e. it is not 221 secreted outside.

222 When considering the phylogenetic tree LOX domain (Figs. 3-6), it is tempting to think that 223 LOX originated from actinomycetes, and then spread to other branches of prokaryotes and 224 finally jumped into the animal kingdom at the very beginning of the branching. At the same 225 time, of course, the loss of lysyl oxidase gene occurred repeatedly, as has happened, for example, 226 in ctenophores and nematodes. But, of course, many other scenarios are possible, since all 227 events of horizontal transfer of LOX are very old. We should mention another fundamental 228 aspect of the project, emphasizing its importance. There is still relatively little research on the 229 influence of horizontal gene transfer with the subsequent adaptation to a new host on the 230 catalytic properties of enzymes.

231 Many questions arise from these facts. For example, what is the origin of the lysyl oxidase in 232 animals? Has it occurred in primitive animals at the beginning of their evolution from bacteria, 233 as often happened? Or, conversely, LOX genes, which have important functions in animals, "ran 234 away" several times in the world of microbes? When this second embodiment is less likely due 235 to disruption of animal into exons, but it allows the most economical way to explain the 236 evolution of the composition of the domain in animals and bacteria. The obvious fact is only that 237 "hops" between the groups of prokaryotes were repeated several times.

238 CONCLUSIONS

239 N-terminally truncated H. turkmenica LOX (HTU-LOX) may be successfully expressed 240 in E. coli 241 Optimal refolding conditions is different from those for the growth of the host cells 242 Sensitivity to β-aminopropionitrile is conserved in HTU-LOX 243 HTU-LOX has a relaxed substrate specificity in comparison with mammalian LOX is of 244 the former 245 Benzyl amine is a poor substrate for HTU-LOX 246 H. turkmenica cells and culture medium do not contain any detectable amine oxidase 247 activity 248 In H. turkmenica, the lysyl oxidase is present mostly as an unprocessed protein 249 Native H. turkmenica lysyl oxidase function may be cryptic

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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 250 ACKNOWLEDGMENTS

251 These studies were supported by MCB RAS and Russian Foundation for Basic Research (grant 252 16-04-01755).

253 REFERENCES

254 Grau-Bove X, Ruiz-Trillo I, Rodriguez-Pascual F (2015). Origin and evolution of lysyl oxidases. 255 Sci. Rep. 5, 10568.

256 Noda-García L, Camacho-Zarco AR, Medina-Ruíz S, Gaytán P, Carrillo-Tripp M, Fülöp V, 257 Barona-Gómez F. (2013) Evolution of substrate specificity in a recipient's enzyme following 258 horizontal gene transfer. Mol Biol Evol. 30, 2024-2034.

259 Saunders E, Tindall BJ, Fähnrich R, Lapidus A, Copeland A, Del Rio TG, Lucas S, Chen F, Tice 260 H, Cheng JF, Han C, Detter JC, Bruce D, Goodwin L, Chain P, Pitluck S, Pati A, Ivanova N, 261 Mavromatis K, Chen A, Palaniappan K, Land M, Hauser L, Chang YJ, Jeffries CD, Brettin T, 262 Rohde M, Göker M, Bristow J, Eisen JA, Markowitz V, Hugenholtz P, Klenk HP, Kyrpides NC. 263 (201) Complete genome sequence of Haloterrigena turkmenica type strain (4k). Stand Genomic 264 Sci 2: 107-116. 265

266 Ventosa A, Carmen Gutierrez M, Kamekura M, Dyall-Smith M L. (1999). Proposal to transfer 267 Halococcus turkmenicus, trapanicum JCM 9743 and strain GSL-11 to 268 Haloterrigena turkmenica gen. nov., comb. nov. Int. J. Syst. Bacter. 49, 131-6.

269 Zvyagintseva IS, Tarasov AL (1987). Extreme halophilic bacteria from alkaline soils. 270 Mikrobiologiya 56, 839-44.

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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 272 FIGURES

273

274 Figure 1. Electrophoretic analysis of the different fractions of proteins 275 HTU-AADN and HTU-QVED, fragments of H. turkmenica LOX. A. 276 molecular weight marker proteins. B, D protein HTU-QVED. C, E protein 277 HTU-AADN.

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292 Figure 2. Western blot of cell lysate (A) and supernatant (B) of the culture medium H. 293 turkmenica. Marker length proteins on the right.

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295 Fig. 3. Phylogenetic tree of the catalytic domain of lysyl oxidase.

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298 lox 299 loxl1 MALAGAGSQLRTLVWSACLCVLVHGQQAQPGQGSDPGRWRQLIQWENNGQVYSLLNSGSE 60 300 Haloterrigena MILNWIKERKRATALVGVLLIVVAGVGMITLGGVAADNPFTV ------20 301 302 303 loxl1 EPPYLPVRSSDAPSQGGERNGAQQGRLSVGSVYRPNQNGRGLPDLVPDPNYVQASTYVQR 420 304 LOX VGDDPYNPYKYSDDNPYYNYYDTYERPRPGSRNRPGYGTGYFQYGLPDLVPDPYYIQASTYVQK 64 305 Haloterrigena SDSSTDTSTTSGSEDTANEEATPVDEENSTTPSTTESDSEPSDDQVEDDQVEDQPEVNFV 102 306 * * * : . 307 308 loxl1 AHLYSLRCAAEEKCLASTAYAPEATDYDLRVLLRFPQRVKNQGTADFLPNRPRHT---WE 477 309 LOX MSMYNLRCAAEENCLASSAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYS---WE 121 310 Haloterrigena PGVRNFDVSIEEFDESSADVEDGFVTPGEHRLLRFDMIIYNVGDADAELGHPENRSDLFE 162 311 : .: : ** :*: . : **** : * * :* .:*. :* 312 313 loxl1 WHSCHQHYHSMDEFSHYDLLDASTGKKVAEGHKASFCLEDSTCDFGNLKR-YACTSHTQG 536 314 LOX WHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRR-FACTAHTQG 180 315 Haloterrigena YSDSHNHAH-LKGFNKYKILDEAG-NEMNAGKKQTFCLRDNFQTRSNASSSAKFDCDYQG 220 316 : ..*:* * :. *.:*.:** ..: *:* :***.*. . .. ** 317 318 loxl1 LSPGCYDTYNADIDCQWIDITDVQPGNYILKVHVNPKYIVLESDFTNNVVRCNIHYTGRY 596 319 LOX LSPGCYDTYAADIDCQWIDITDVQPGNYILKVSVNPSYLVPESDYTNNVVRCDIRYTGHH 240 320 Haloterrigena ISAGWADVYPASLPGQYLVIDDLPDGEYTLQATTNAEGTIDEKCDDDNTVRVDLRINNDT 280 321 :*.* *.* *.: *:: * *: *:* *:. .*.. : *. :*.** ::: .. 322 323 loxl1 VSTTNCKIVQS----- 607 324 LOX AYASGCTISPY----- 251 325 Haloterrigena VTVHSSQDDYVKPPSC 296 326

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329 Fig. 4. Alignment of amino acid sequences of the mouse lysyl oxidase (LOXL1 and LOX isoforms) and 330 Haloterrigena turkmenica (Haloterrigena). Cysteine residues marked in yellow marked, LTQ-forming 331 lysine and tyrosine - red, , purple - hyperconserved three histidine residues necessary for the binding of 332 copper (the whole site is underlined). Dark green – hypothetical proteolytic cleavage sites.

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334

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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 335 336 HaloterrigenalimiCola MKIKRIKGRKRAAALIGVFLIVVAGIGIITLGGVAVDNPFTVSDRSTDTPTTSE-SEGTT 337 HaloterrigenaturCk MILNWIKERKRATALVGVLLIVVAGVGMITLGGVAADNPFTVSDSSTDTSTTSG-SEDTA 338 NatronoCoCCusjeotgali -----MKDRKRATVLVGIILIAVVGAGIITLGDVTVNNPFTVNDSNTDTSTTSE-SEGTA 339 NitrosopumilussalariaBD31 ------MFAAPMIMDAAAAKGGNGNGNNNGGN 340 Nitrosopumilus --MTYTKKIFRKTTLIPVLLAI------GFMFTTPMLLDVAAAPGGNGNG-NGGST 341 *: :. : . . . . 342 343 HaloterrigenalimiCola DEGATPADEENAATPPPTTVESDPKPS-----DDHVEDRTGVNFVPGVENFNVSTEVFDE 344 HaloterrigenaturCk NEEATPVDEENSTT-PST-TESDSEPSDDQVEDDQVEDQPEVNFVPGVRNFDVSIEEFDE 345 NatronoCoCCusjeotgali NEEATPVDKENPAT-SSTPAESNSEPS-----DAQVEDKPEVNFVPGVRDFSISTEEFDE 346 NitrosopumilussalariaBD31 DETTIPTNALLPDVSPGVPKHLNIHNQ------QQKEFLRFTNVWANLGPGTLEFEP 347 Nitrosopumilus ---SIPSDALLPDISPGVPKHLNIHNQ------QQNEFLRFTNTWNNVGVGALEFEP 348 : * : . . . : . ::. :.*. :.. . *: 349 350 HaloterrigenalimiCola --SSPDVEDGFVTPGEHRLLRFDMIIYNMGDADAELGRPENR-----PDLFEYSESHGHA 351 HaloterrigenaturCk --SSADVEDGFVTPGEHRLLRFDMIIYNVGDADAELGHPENR-----SDLFEYSDSHNHA 352 NatronoCoCCusjeotgali --SSTDVEDGFVTPGEHRLLRFDTIIYNLGDADAELGHPENR-----SDQFEYSDSHNHA 353 NitrosopumilussalariaBD31 LFPDPDADEGTTQDA------FQNLYDDEGNFGLTDQNVWHENVSQFIFHEAHNHW 354 Nitrosopumilus VFPDSDAVEGTTQDA------FQNLYDDAGNFAIPSQKIWSTTVSEFIFHETHNHW 355 ...*. :* . . : *: * .::. ..:. . * : ::*.* 356 357 HaloterrigenalimiCola HLKGFNNYILL-DESGE------RTGAVRKQTFCLRDLYQTRSTASSSPQ---FDC 358 HaloterrigenaturCk HLKGFNKYKIL-DEAGN------EMNAGKKQTFCLRDNFQTRSNASSSAK---FDC 359 NatronoCoCCusjeotgali HLKGFNKYALF-DESGN------EMDMGKKQTFCLRDDFQTRSNASSSAK---FNC 360 NitrosopumilussalariaBD31 HIDNVGEFAVRAYDPNNPDVPGDIV--DDAASIKVGFCITNVFKYNGEESPTSQRIYWDC 361 Nitrosopumilus HISDIGEFSIRSDDNGVPGEIAKNVNGDDVAAVKVGFCIADVYKYNGDNSPTSQRVYWDC 362 *:....:: : : . * **: : :: .. *.:.: ::* 363 364 HaloterrigenalimiCola E--YQGISAGWADEYDASLPGQYIVIDDLPDGEYTLQATTNAAGTI--NETCDGDNTVRV 365 HaloterrigenaturCk D--YQGISAGWADVYPASLPGQYLVIDDLPDGEYTLQATTNAEGTI--DEKCDDDNTVRV 366 NatronoCoCCusjeotgali D--YQGISAGWADVYPASLPGQYLVIDGLPDGEYTLHATTNAEGTI--DEKCDDDNTVRV 367 NitrosopumilussalariaBD31 EVGLQGIQPGWVDQYHQSVEGNEINITKVPNGTYFLTHTWNPANAFVDADNSNNVSWMKF 368 Nitrosopumilus EVGLQGIAPGWADQYHQSVEGNEINITDLPNGTYFLVHKWNPANAFVDADVSNDESWMKF 369 : *** .**.* * *: *: : * :*:* * * . *. .:: : .:. . :.. 370 371 HaloterrigenalimiCola DL------SINNDTVTVHTPQSHYVRPSAC------372 HaloterrigenaturCk DL------RINNDTVTVHSSQDDYVKPPSC------373 NatronoCoCCusjeotgali DL------RINNDTVTVLSSQEDHVKPSAC------374 NitrosopumilussalariaBD31 ELTDDGNGNRKINEIEGFAPECQDDDSTPGICGDINKNS 375 Nitrosopumilus DLTDDGNGNRKIVEIEGFAPECQGDGSTPGICGEINKNN 376 :* * : . * * * 377 378 Fig. 5. Multiple alignment of archeal lysyl oxidases. (HaloterrigenaturCk – Haloterrigena 379 turkmenica, NatronoCoCCusjeotgali – Natronococcus jeotgali, HaloterrigenalimiCola – 380 Haloterrigena limicola, Nitrosopumilus – Nitrosopumilus. 381 382 383 384 385 386 387 388

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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

389 ARCHEA ------KESNSEPSDDQVEDKPEVNFVPGVRNFGVSTEEFDESSPDVEDGFVTPGEHRL 390 DELTA ------VDADVISRTVYIERRTFAADACEVYEGCVGAPGR------RRL 391 FUNGI ------DADWLQKHLYIDYVDAAEDPCLINEGCLTGPPA------392 ANIMA ------MDALLVQQTAHLEDRPLYLLGCAMEENCLASSAYQVEPGWPYGTRRL 393 LOW ------MNSNNAQSTLVLSAGHLYNTQCAMEEGCLASGAW------RKL 394 BACIES ------QCPPGTNCELLPDLVILPRFTRSQIKEYSNDDPY------YGGQ 395 short ------PNRLLPDLVIYPPSELSIVGSEKTG------RRE 396 BETA ------ATTNRLPNLKPLPASNLSLVADSAGGST------397 ACTINshort ------LPDLRQAPIGDLQVQTG-----PS------GQVR 398 ACTINvar ------AK------AVR 399 STREPTOM QAPAPALKANAKRPTKATVPNVPKPDLRSLPAYGITVSDGYEDVPG------KDY 400 401 402 ARCHEA LRFDMIIYNLG-DADAELGRPE------N 403 DELTA LRFSVSIPNLG-SAAVIPPPE------E 404 FUNGI ------DRDDP------N 405 ANIMA LRFTARIWNRG-TADFLPK------R 406 LOW LRFSASFWNFG-TADFLP------N 407 BACIES LRFAATIANIG-DGPMETRGYCGTLGVVSNSICPDGSYPRQVLFQRIYSLKDKNLSSVDR 408 short IKFATTVWNIGKSGPLELIGTV------DPATNKTRVYQRIKNRGGESAS---R 409 BETA LRFNTTSWNKG-SGPLVLGAGA------VDTGSGKQQVFQRVFLSNGGYFV---P 410 ACTINshort LRFTTSIVNV--DGPLLLVAHR------DSTDVPFMAVQAI-QSDGSIADV--E 411 ACTINvar LRFTAAEWNAG-DGPLLLYGRR------DSATDTMDVRQYFFDAKHGQVQR--Q 412 STREPTOM LAFSANVWNAG-PAKLVVDGFR------SPGKELMDAYQYFYDAKGKQVGY--T 413 414 415 ARCHEA RPDQFEYSES--HGHAH-LKGFNKYAI--LDESGN---EMNAGKKQTFCLRDVFQTRS-- 416 DELTA NPDLYVYDEC--HQHEH-LVNFASYEL--RDADNN---VVAVGRKQGFYLVD-MEPYC-- 417 FUNGI NPPYWHWDTC--HEHWH-FTAYANYRL--LSANGS--EVVAQGHKNGFCLED-SLCDE-- 418 ANIMA PRHSWEWHAC--HQHYHSMEVFAHYDL--LDLNGT---KVAEGHKASFCLED-TECDG-- 419 LOW PDDGPEWHEC--HNHYH-ISNFANYTI--TGSAGN---QLTQGHKQSFCLED-VKCLP-- 420 BACIES PAGTNYYDNQPGHNHYH-VDDWVEFRLVKIEP-GKRASIIAKGRKVSYCLFDSGICNNAD 421 short TAGYFEYHPD--HEHWHLFNDFATYELWTLNADGSLETLVATSGKVTFCLMDTTAVDP-- 422 BETA VAGGFEWHPA--HNHFH-FDDFALYTLQPVNAPGG---VVRTGSKTTFCLMDSTRIDS-- 423 ACTINshort TPASLYYEPADGHDHWH-LLDFEYYQL--RRPDGG---VVVTDRKNGFCIGDRYRVRD-- 424 ACTINvar TAGTMYYEPAPMHQHWH-LLDFARYQL--RTPDGE---TVVRDRKNGFCLADRYADVD-- 425 STREPTOM PTGTMEWDPRPGHEHWH-FTDFASYRL--LKADKK---ETVRSGKEAFCLANTDAVD--- 426 : * * * . : : : . * : : : 427 428 ARCHEA ------NASSS------AKFDCE------YQGISAGWADVYP-ASLPGQYLVI-D 429 DELTA ------DAAPR------AYTCG------GQGISPGWSDTYA-ADTPCQWLDV-T 430 FUNGI ------GVAP------FYNCT------NQGITMGCHDLYD-AGLGCQWIDI-T 431 ANIMA ------GVQRR------YCANYG------DQGISVNCWDTYR-HDIDCQWIDI-T 432 LOW ------SLLPK------YICN------NQGISVGCADSYSVSNIDCQWIDI-T 433 BACIES SLCTINGTVYGERNLSNYGLGNYASCN-----AMKQGISVGGYDTYG-VMYEGQFLQLPK 434 short ------YPLPN---APGGP--TYSSCG-----NNVQGISVGWGDTYG-AKLAGQEIDL-T 435 BETA ------SLPG---APGQA--VYSTCG-----RTIGGISVGWGDTYG-AHLPGQEIDF-T 436 ACTINshort ------DLPG---RPADPYVLGHMCG-PAALTVKMGISVGWGDDYK-HTLPFQWLDI-T 437 ACTINvar ------YTLPNAVWRPENT-DLATSCGDPSSLDVREGISVGS-DDYR-YTVDFQWLDI-T 438 STREPTOM ------YTVKNANWHPDNT-DLSTACGQENSISVREVLDVGSGDTYT-QDLPGQSFDI-T 439 : . * * * : . 440 441 ARCHEA DL------PDGEYTLQATTNAEGTIDEKCDDDNTVRVDLRI------442 DELTA DV------PDGTYTLRVGVDTRDIVDEGDVHPNTVDVPVR------443 FUNGI DLHLQPGYSPNTEYTLSVILNPEKAIPETDYSNNAAV------444 ANIMA DV------PPGNYILKVVVNPEFAVAESDFTNNAVRCNIRY------445 LOW PL------KSGWYVLNVYVNPDKRVTESDYTNNVFHVLFRF------446 BACIES GL------ASGTYILEIE-DPTGSFYEKNRSNNLFRMPIVIEKQ------447 short DV------PDGRYLLRVEVDPEDRIEELDYDNNFSVTFVEI------448 BETA GN------ADGTYQLRIVIDPNKVIIESDESNNASCVLISIRKPNTVTVLDSSGSCSTA 449 ACTINshort GL------PAGRYDLVNVADPDGALLEKNYDNNASWVDISVTSP------450 ACTINvar HV------PSGTYDLVNTVNPDRTL-ETSYDNNSSSIAIVLGGT------451 STREPTOM GL------PNGTYYIQVLANPENRLKETNHKNNSALRKVVLGGK------452 * : :. . * * 453 Fig. 6. Multiple alignment of the catalytic domains from all lysyl oxidases representing different branches 454 of Life.

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PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2691v1 | CC BY 4.0 Open Access | rec: 3 Jan 2017, publ: 3 Jan 2017