{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T13:45:43Z","timestamp":1777643143163,"version":"3.51.4"},"reference-count":42,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2017,3,15]],"date-time":"2017-03-15T00:00:00Z","timestamp":1489536000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2017,3,15]],"date-time":"2017-03-15T00:00:00Z","timestamp":1489536000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>(+)-\u03b3-lactamase catalyzes the specific hydrolysis of (+)-\u03b3-lactam out of the racemic \u03b3-lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one) to leave optically pure (\u2212)-\u03b3-lactam, which is the key building block of antiviral drugs such as carbovir and abacavir. However, no structural data has been reported on how the enzymes bind the \u03b3-lactams and achieve their enantioselectivities. We previously identified an isochorismatase-like hydrolase (IHL, Mh33H4-5540) with (+)-\u03b3-lactamase activity, which constitutes a novel family of \u03b3-lactamase. Here, we first discovered that this enzyme actually hydrolyzed both (+)- and (\u2212)-\u03b3-lactam, but with apparently different specificities. We determined the crystal structures of the apo-form, (+)-\u03b3-lactam bound, and (\u2212)-\u03b3-lactam bound forms of the enzyme. The structures showed that the binding sites of both (+) and (\u2212)-\u03b3-lactam resemble those of IHLs, but the \u201ccover\u201d loop conserved in IHLs is lacking in the enzyme, probably resulting in its incomplete enantioselectivity. Structural, biochemical, and molecular dynamics simulation studies demonstrated that the steric clash caused by the binding-site residues, especially the side-chain of Cys111 would reduce the binding affinity of (\u2212)-\u03b3-lactam and possibly the catalytic efficiency, which might explain the different catalytic specificities of the enantiomers of \u03b3-lactam. Our results would facilitate the directed evolution and application of Mh33H4-5540 in antiviral drug synthesis.<\/jats:p>","DOI":"10.1038\/srep44542","type":"journal-article","created":{"date-parts":[[2017,3,15]],"date-time":"2017-03-15T13:03:35Z","timestamp":1489583015000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Structural insights into the \u03b3-lactamase activity and substrate enantioselectivity of an isochorismatase-like hydrolase from Microbacterium hydrocarbonoxydans"],"prefix":"10.1038","volume":"7","author":[{"given":"Shuaihua","family":"Gao","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yu","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weiwei","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wenhe","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"You","family":"Yu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yajuan","family":"Mu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hao","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xinqi","family":"Gong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guojun","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yue","family":"Feng","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2017,3,15]]},"reference":[{"key":"BFsrep44542_CR1","doi-asserted-by":"publisher","first-page":"2381","DOI":"10.1016\/0957-4166(96)00293-5","volume":"7","author":"N Hiroto","year":"1996","unstructured":"Hiroto, N., Kazuto, I., Yuko, O. & Hiroshi, H. Lipase-catalyzed resolution of 2-azabicyclo[2.2.1]hept-5-en-3-ones. Tetrahedron: Asymmetry 7, 2381\u20132386 (1996).","journal-title":"Tetrahedron: Asymmetry"},{"key":"BFsrep44542_CR2","doi-asserted-by":"publisher","first-page":"4642","DOI":"10.1021\/cr2004822","volume":"112","author":"R Singh","year":"2012","unstructured":"Singh, R. & Vince, R. 2-Azabicyclo[2.2.1]hept-5-en-3-one: chemical profile of a versatile synthetic building block and its impact on the development of therapeutics. Chem. Rev. 112, 4642\u20134686 (2012).","journal-title":"Chem. Rev."},{"key":"BFsrep44542_CR3","doi-asserted-by":"crossref","unstructured":"Taylor, S. J. C. et al. Chemoenzymatic synthesis of (\u2212)-carbovir utilizing a whole cell catalysed resolution of 2-azabicyclo[2.2.1]hept-5-en-3-one. J. Chem. Soc., Chem. Commun. 1120\u20131121 (1990).","DOI":"10.1039\/C39900001120"},{"key":"BFsrep44542_CR4","doi-asserted-by":"publisher","first-page":"1117","DOI":"10.1016\/S0957-4166(00)80218-9","volume":"4","author":"SJC Taylor","year":"1993","unstructured":"Taylor, S. J. C. et al. Development of the biocataIytic resolution of 2-azabicycloI2,2,l]hept-5-en-3-one as an entry to single-emmtiomer carbocyclic nucleosides. Tetrahedron: Asymmetry 4, 1117\u20131128 (1993).","journal-title":"Tetrahedron: Asymmetry"},{"key":"BFsrep44542_CR5","doi-asserted-by":"publisher","first-page":"2163","DOI":"10.1016\/S0968-0896(99)00146-7","volume":"7","author":"JCT Stephen","year":"1999","unstructured":"Stephen, J. C. T., Rob, C. B., Phil, A. K. & Ian, N. T. Novel screening methods -the key to cloning commercially successful biocatalysts. Bioorg. Med. Chem. Lett. 7, 2163\u20132168 (1999).","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"BFsrep44542_CR6","doi-asserted-by":"publisher","first-page":"519","DOI":"10.1016\/j.jmb.2004.03.001","volume":"338","author":"K Line","year":"2004","unstructured":"Line, K., Isupov, M. N. & Littlechild, J. A. The crystal structure of a (\u2212) gamma-lactamase from an Aureobacterium species reveals a tetrahedral intermediate in the active site. J. Mol. Biol. 338, 519\u2013532 (2004).","journal-title":"J. Mol. Biol."},{"key":"BFsrep44542_CR7","doi-asserted-by":"publisher","first-page":"711","DOI":"10.1016\/j.tet.2003.11.064","volume":"60","author":"HS Toogood","year":"2004","unstructured":"Toogood, H. S. et al. The use of a thermostable signature amidase in the resolution of the bicyclic synthon (rac)-gamma-lactam. Tetrahedron 60, 711\u2013716 (2004).","journal-title":"Tetrahedron"},{"key":"BFsrep44542_CR8","doi-asserted-by":"publisher","first-page":"7492","DOI":"10.1128\/AEM.01398-12","volume":"78","author":"S Zhu","year":"2012","unstructured":"Zhu, S., Gong, C., Song, D., Gao, S. & Zheng, G. Discovery of a novel (+)-gamma-lactamase from Bradyrhizobium japonicum USDA 6 by rational genome mining. Appl. Environ. Microbiol. 78, 7492\u20137495 (2012).","journal-title":"Appl. Environ. Microbiol."},{"key":"BFsrep44542_CR9","doi-asserted-by":"publisher","first-page":"4899","DOI":"10.1016\/j.bmcl.2014.08.057","volume":"24","author":"SZ Zhu","year":"2014","unstructured":"Zhu, S. Z. et al. Enzymatic preparation of optically pure (+)-2-azabicyclo[2.2.1]hept-5-en-3-one by (\u2212)-gamma-lactamase from Bradyrhizobium japonicum USDA 6. Bioorg. Med. Chem. Lett. 24, 4899\u20134902 (2014).","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"BFsrep44542_CR10","doi-asserted-by":"publisher","first-page":"3878","DOI":"10.1016\/j.bmcl.2015.07.054","volume":"25","author":"SH Gao","year":"2015","unstructured":"Gao, S. H., Zhu, S. Z., Huang, R., Lu, Y. X. & Zheng, G. J. Efficient synthesis of the intermediate of abacavir and carbovir using a novel (+)-gamma-lactamase as a catalyst. Bioorg. Med. Chem. Lett. 25, 3878\u20133881 (2015).","journal-title":"Bioorg. Med. Chem. Lett."},{"key":"BFsrep44542_CR11","doi-asserted-by":"publisher","first-page":"170","DOI":"10.1007\/s12010-015-1565-7","volume":"176","author":"L Ren","year":"2015","unstructured":"Ren, L., Zhu, S., Shi, Y., Gao, S. & Zheng, G. Enantioselective resolution of gamma-lactam by a novel thermostable type II (+)-gamma-lactamase from the hyperthermophilic archaeon Aeropyrum pernix. Appl. Biochem. Biotechnol. 176, 170\u2013184 (2015).","journal-title":"Appl. Biochem. Biotechnol."},{"key":"BFsrep44542_CR12","doi-asserted-by":"publisher","first-page":"3069","DOI":"10.1007\/s00253-014-6114-8","volume":"99","author":"J Wang","year":"2015","unstructured":"Wang, J., Zhu, Y., Zhao, G., Zhu, J. & Wu, S. Characterization of a recombinant (+)-gamma-lactamase from Microbacterium hydrocarbonoxydans which provides evidence that two enantiocomplementary gamma-lactamases are in the strain. Appl. Microbiol. Biotechnol. 99, 3069\u20133080 (2015).","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"BFsrep44542_CR13","doi-asserted-by":"publisher","first-page":"4691","DOI":"10.1007\/s00253-014-6247-9","volume":"99","author":"JJ Wang","year":"2015","unstructured":"Wang, J. J., Zhu, J. G. & Wu, S. Immobilization on macroporous resin makes E-coli RutB a robust catalyst for production of (\u2212) Vince lactam. Appl. Microbiol. Biotechnol. 99, 4691\u20134700 (2015).","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"BFsrep44542_CR14","doi-asserted-by":"crossref","unstructured":"Zhu, S., Huang, R., Gao, S., Li, X. & Zheng, G. Discovery and characterization of a second extremely thermostable (+)-gamma-lactamase from Sulfolobus solfataricus P2. J. Biosci. Bioeng. (2015).","DOI":"10.1016\/j.jbiosc.2015.09.012"},{"key":"BFsrep44542_CR15","doi-asserted-by":"publisher","first-page":"2517","DOI":"10.1002\/cctc.201402077","volume":"6","author":"Z Assaf","year":"2014","unstructured":"Assaf, Z. et al. Identification and Application of Enantiocomplementary Lactamases for Vince Lactam Derivatives. ChemCatChem 6, 2517\u20132521 (2014).","journal-title":"ChemCatChem"},{"key":"BFsrep44542_CR16","doi-asserted-by":"publisher","first-page":"209","DOI":"10.2174\/1389203003381405","volume":"1","author":"M Holmquist","year":"2000","unstructured":"Holmquist, M. Alpha\/Beta-hydrolase fold enzymes: structures, functions and mechanisms. Curr Protein Pept Sci 1, 209\u2013235 (2000).","journal-title":"Curr Protein Pept Sci"},{"key":"BFsrep44542_CR17","doi-asserted-by":"publisher","first-page":"284","DOI":"10.1107\/S0907444900016838","volume":"57","author":"IS Gonsalvez","year":"2001","unstructured":"Gonsalvez, I. S., Isupov, M. N. & Littlechild, J. A. Crystallization and preliminary X-ray analysis of a gamma-lactamase. Acta Crystallogr D Biol Crystallogr 57, 284\u2013286 (2001).","journal-title":"Acta Crystallogr D Biol Crystallogr"},{"key":"BFsrep44542_CR18","doi-asserted-by":"crossref","unstructured":"Gao, S., Huang, R., Zhu, S., Li, H. & Zheng, G. Identification and characterization of a novel (+)-gamma-lactamase from Microbacterium hydrocarbonoxydans. Appl Microbiol Biotechnol (2016).","DOI":"10.1007\/s00253-016-7643-0"},{"key":"BFsrep44542_CR19","doi-asserted-by":"publisher","first-page":"2887","DOI":"10.1110\/ps.051783005","volume":"14","author":"J Caruthers","year":"2005","unstructured":"Caruthers, J. et al. Crystal structures and proposed structural\/functional classification of three protozoan proteins from the isochorismatase superfamily. Protein Sci 14, 2887\u20132894 (2005).","journal-title":"Protein Sci"},{"key":"BFsrep44542_CR20","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1007\/s10969-012-9127-5","volume":"13","author":"AM Goral","year":"2012","unstructured":"Goral, A. M. et al. Crystal structure of a putative isochorismatase hydrolase from Oleispira antarctica. J Struct Funct Genomics 13, 27\u201336 (2012).","journal-title":"J Struct Funct Genomics"},{"key":"BFsrep44542_CR21","doi-asserted-by":"publisher","first-page":"5684","DOI":"10.1021\/bi027385d","volume":"42","author":"JF Parsons","year":"2003","unstructured":"Parsons, J. F., Calabrese, K., Eisenstein, E. & Ladner, J. E. Structure and mechanism of Pseudomonas aeruginosa PhzD, an isochorismatase from the phenazine biosynthetic pathway. Biochemistry 42, 5684\u20135693 (2003).","journal-title":"Biochemistry"},{"key":"BFsrep44542_CR22","doi-asserted-by":"publisher","first-page":"1687","DOI":"10.1007\/s12010-015-1670-7","volume":"176","author":"TY Xue","year":"2015","unstructured":"Xue, T. Y., Xu, G. C., Han, R. Z. & Ni, Y. Soluble Expression of (+)-gamma-Lactamase in Bacillus subtilis for the Enantioselective Preparation of Abacavir Precursor. Appl Biochem Biotechnol 176, 1687\u20131699 (2015).","journal-title":"Appl Biochem Biotechnol"},{"key":"BFsrep44542_CR23","doi-asserted-by":"publisher","first-page":"774","DOI":"10.1016\/j.jmb.2007.05.022","volume":"372","author":"E Krissinel","year":"2007","unstructured":"Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J Mol Biol 372, 774\u2013797 (2007).","journal-title":"J Mol Biol"},{"key":"BFsrep44542_CR24","doi-asserted-by":"publisher","first-page":"W545","DOI":"10.1093\/nar\/gkq366","volume":"38","author":"L Holm","year":"2010","unstructured":"Holm, L. & Rosenstrom, P. Dali server: conservation mapping in 3D. Nucleic Acids Res 38, W545\u2013549 (2010).","journal-title":"Nucleic Acids Res"},{"key":"BFsrep44542_CR25","first-page":"536","volume":"247","author":"AG Murzin","year":"1995","unstructured":"Murzin, A. G., Brenner, S. E., Hubbard, T. & Chothia, C. SCOP: a structural classification of proteins database for the investigation of sequences and structures. J Mol Biol 247, 536\u2013540 (1995).","journal-title":"J Mol Biol"},{"key":"BFsrep44542_CR26","doi-asserted-by":"publisher","first-page":"9176","DOI":"10.1002\/anie.200903407","volume":"48","author":"PK Fyfe","year":"2009","unstructured":"Fyfe, P. K., Rao, V. A., Zemla, A., Cameron, S. & Hunter, W. N. Specificity and mechanism of Acinetobacter baumanii nicotinamidase; implications for activation of the front line TB drug pyrazinamide. Angewandte Chemie 48, 9176\u20139179 (2009).","journal-title":"Angewandte Chemie"},{"key":"BFsrep44542_CR27","doi-asserted-by":"publisher","first-page":"269","DOI":"10.1006\/jmbi.1996.0574","volume":"263","author":"A Zajc","year":"1996","unstructured":"Zajc, A., Romao, M. J., Turk, B. & Huber, R. Crystallographic and fluorescence studies of ligand binding to N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. J Mol Biol 263, 269\u2013283 (1996).","journal-title":"J Mol Biol"},{"key":"BFsrep44542_CR28","doi-asserted-by":"publisher","first-page":"16842","DOI":"10.1074\/jbc.M602294200","volume":"281","author":"Y Hamano","year":"2006","unstructured":"Hamano, Y., Matsuura, N., Kitamura, M. & Takagi, H. A novel enzyme conferring streptothricin resistance alters the toxicity of streptothricin D from broad-spectrum to bacteria-specific. J Biol Chem 281, 16842\u201316848 (2006).","journal-title":"J Biol Chem"},{"key":"BFsrep44542_CR29","doi-asserted-by":"publisher","first-page":"307","DOI":"10.1016\/S0076-6879(97)76066-X","volume":"276","author":"Z Otwinowski","year":"1997","unstructured":"Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307\u2013326 (1997).","journal-title":"Methods Enzymol"},{"key":"BFsrep44542_CR30","doi-asserted-by":"publisher","first-page":"235","DOI":"10.1107\/S0907444910045749","volume":"67","author":"MD Winn","year":"2011","unstructured":"Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235\u2013242 (2011).","journal-title":"Acta Crystallogr. D"},{"key":"BFsrep44542_CR31","doi-asserted-by":"publisher","first-page":"582","DOI":"10.1107\/S0907444909012098","volume":"65","author":"TC Terwilliger","year":"2009","unstructured":"Terwilliger, T. C. et al. Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizard. Acta Crystallogr. D 65, 582\u2013601 (2009).","journal-title":"Acta Crystallogr. D"},{"key":"BFsrep44542_CR32","doi-asserted-by":"publisher","first-page":"2126","DOI":"10.1107\/S0907444904019158","volume":"60","author":"P Emsley","year":"2004","unstructured":"Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126\u20132132 (2004).","journal-title":"Acta Crystallogr D Biol Crystallogr"},{"key":"BFsrep44542_CR33","doi-asserted-by":"publisher","first-page":"1948","DOI":"10.1107\/S0907444902016657","volume":"58","author":"PD Adams","year":"2002","unstructured":"Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 58, 1948\u20131954 (2002).","journal-title":"Acta Crystallogr D Biol Crystallogr"},{"key":"BFsrep44542_CR34","unstructured":"WL, D. The PyMOL molecular graphics system (Delano Scientific, San Carlos, CA) (2002)."},{"key":"BFsrep44542_CR35","doi-asserted-by":"publisher","first-page":"351","DOI":"10.1021\/jp021564n","volume":"106","author":"MA Jacobson","year":"2002","unstructured":"Jacobson, M. A., Kaminski, G. A., Friesner, G. A. & Rapp, C. S. Force field validation using protein side chain prediction. J Phys Chem B 106, 351\u2013367 (2002).","journal-title":"J Phys Chem B"},{"key":"BFsrep44542_CR36","doi-asserted-by":"publisher","first-page":"351","DOI":"10.1002\/prot.10613","volume":"55","author":"MP Jacobson","year":"2004","unstructured":"Jacobson, M. P. et al. A hierarchical approach to all-atom protein loop prediction. Proteins 55, 351\u2013367 (2004).","journal-title":"Proteins"},{"key":"BFsrep44542_CR37","doi-asserted-by":"publisher","first-page":"368","DOI":"10.1002\/prot.20014","volume":"55","author":"X Li","year":"2004","unstructured":"Li, X., Jacobson, M. P. & Friesner, R. A. High-resolution prediction of protein helix positions and orientations. Proteins 55, 368\u2013382 (2004).","journal-title":"Proteins"},{"key":"BFsrep44542_CR38","unstructured":"AMBER 10 (University of California,San Francisco, 2008)."},{"key":"BFsrep44542_CR39","doi-asserted-by":"publisher","first-page":"1049","DOI":"10.1002\/1096-987X(200009)21:12<1049::AID-JCC3>3.0.CO;2-F","volume":"21","author":"J Wang","year":"2000","unstructured":"Wang, J., Cieplak, P. & Kollman, P. A. How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? Journal of computational chemistry 21, 1049\u20131074 (2000).","journal-title":"Journal of computational chemistry"},{"key":"BFsrep44542_CR40","doi-asserted-by":"publisher","first-page":"1157","DOI":"10.1002\/jcc.20035","volume":"25","author":"J Wang","year":"2004","unstructured":"Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. Journal of computational chemistry 25, 1157\u20131174 (2004).","journal-title":"Journal of computational chemistry"},{"key":"BFsrep44542_CR41","doi-asserted-by":"publisher","first-page":"3684","DOI":"10.1063\/1.448118","volume":"81","author":"HJ Berendsen","year":"1984","unstructured":"Berendsen, H. J., Postma, J. v., van Gunsteren, W. F., DiNola, A. & Haak, J. Molecular dynamics with coupling to an external bath. The Journal of chemical physics 81, 3684\u20133690 (1984).","journal-title":"The Journal of chemical physics"},{"key":"BFsrep44542_CR42","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1016\/0021-9991(83)90014-1","volume":"52","author":"HC Andersen","year":"1983","unstructured":"Andersen, H. C. RATTLE: A \u201cVelocity\u201d version of the SHAKE algorithm for molecular dynamics calculations. Journal of Computational Physics 52, 24\u201334 (1983).","journal-title":"Journal of Computational Physics"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/srep44542.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/srep44542","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/srep44542.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,24]],"date-time":"2022-12-24T00:17:57Z","timestamp":1671841077000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/srep44542"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,3,15]]},"references-count":42,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2017,4,20]]}},"alternative-id":["BFsrep44542"],"URL":"https:\/\/doi.org\/10.1038\/srep44542","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,3,15]]},"assertion":[{"value":"28 September 2016","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"24 January 2017","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"15 March 2017","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing financial interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"44542"}}