Structural Insights into Mycoplasma hyopneumoniae (Mhp) Lipoate Protein Ligase A (LplA)

Mycoplasma hyopneumoniae is a fundamental and prevalent etiologic pathogen of enzootic pneumonia. Although M. hyopneumoniae infection is normally not fatal, it usually leads to respiratory syndrome and secondary infection, thus provoking increased morbidity, mortality, and decreased porcine production worldwide. Disease management strategies such as vaccination, antibiotics, and facility updates can be either costly or ineffectively. To find out novel therapeutical potentials against M. hyopneumoniae infection, the lipoylation system which plays an indispensable role in pathogen energy metabolism, was highlighted.

Lipoylation in M. hyopneumoniae depends greatly on the uptake of lipoic acid from the host, which is mediated by lipoate protein ligase A (LplA). The loss of LplA will lead to the inhibition of pathogen growth and largely decreased virulence. Hence, it is proposed that LplA inhibition will disrupt the lipoylation of energy metabolism-related complexes, thus inhibiting pathogen growth and virulence, laying foundation for novel therapeutic strategies against M. hyopneumoniae infection. More importantly, inhibition of conserved LplA among various pathogens should contribute to a broad spectrum anbiotic effect during infection therapy.

However, the 3D structures and molecular mechanisms of M. hyopneumoniae LplA remain elusive. Here, protein crystallography study of LplA and its substrate H protein, combined with biochemical assays, were performed to provide atomic resolution structure for drug design, and to unveil molecular mechanisms of LplA- mediated lipoylation.

LplA-mediated lipoylation is completed by two steps, namely the lipoate adenylation step and lipoate transfer step. Protein crystallography studies revealed the overall structure of LplA complexed with an intermediate lipoyl-AMP, indicating conserved NTD and CTD connected by a polypeptide motif. The catalytic pocket enclosing lipoyl-AMP lies in the NTD, consistent with the enzymatic assay that the NTD could mediate lipoylation reaction without CTD.

AlphaFold2 predicted the LplA·H protein complex structure, and enzymatic assays identified the acceptor lysine residue in H protein, namely K56, in an inserted loop adjacent to LplA’s catalytic center. The catalytic center displays intensive hydrophobic and polar interactions between lipoyl-AMP and LplA residues, among which K138 and G78 form hydrogen bonds with C10 of lipoyl- AMP, activating the positive charge of this atom, thus facilitating nucleophilic attack of the carbenium ion by the amino group of the H protein K56, bringing the lipoate moiety to the K56 residue.

Structure alignments of LplA against other Lpls implicated a highly conserved lipoylation mechanism across archaea, prokaryotes, and eukaryotes. The almost identical catalytic centers of M. hyopneumoniae LplA and bovine lipotransferase require selective inhibition of pathogen LplA without affecting the host counterpart during infection therapy. Considering that the lipoate adenylation step is much faster than the lipoate transfer step, and given the dominance of the LplA·lipoyl- AMP complex during protein expression, it is suggested that pathogen LplA be inhibited at the lipoate adenylation step to achieve maximum inhibition specificity and minimum side effects.

Overall, this study has presented the high-resolution M. hyopneumoniae LplA structure and detailed molecular mechanisms of LplA-mediated lipoylation, providing fundamental knowledge for potential therapeutic strategies against M. hyopneumoniae infection.
(Word count: 486)

[Ackermann, M. R., Boes, K. M., Brannick, E. M., Breshears, M. A., Brown, D. L., Carlson, C. S., . . . Zachary, J. F. (2017). Contributors. In J. F. Zachary (Ed.), Pathologic Basis of Veterinary Disease (Sixth Edition) (pp. v-vi): Mosby.Barile, M. F., Schimke, R. T., & Riggs, D. B. (1966). Presence of the arginine dihydrolase pathway in Mycoplasma. J Bacteriol, 91(1), 189-192. doi:10.1128/jb.91.1.189-192.1966Beuckelaere, L., Haspeslagh, M., Biebaut, E., Boyen, F., Haesebrouck, F., Krejci, R., . . . Maes, D. (2022). Different local, innate and adaptive immune responses are induced by two commercial Mycoplasma hyopneumoniae bacterins and an adjuvant alone. Front Immunol, 13, 1015525. doi:10.3389/fimmu.2022.1015525Billones, J., Carrillo, M., Organo, V., Macalino, S. J., Emnacen, I., & Bernadette, J. (2013). Virtual Screening Against Mycobacterium tuberculosisLipoate Protein Ligase B (MtbLipB)and In SilicoADMETEvaluation of Top Hits. Oriental Journal of Chemistry, 29(4), 1457-1468. doi:10.13005/ojc/290423Blanchard, B., Vena, M. M., Cavalier, A., Lannic, J. L., Gouranton, J., & Kobisch, M. (1992). Electron microscopic observation of the respiratory tract of SPF piglets inoculated with Mycoplasma hyopneumoniae. Vet Microbiol, 30(4), 329-341. doi:https://doi.org/10.1016/0378-1135(92)90020-TBorziak, K., Posner, M. G., Upadhyay, A., Danson, M. J., Bagby, S., & Dorus, S. (2014). Comparative Genomic Analysis Reveals 2-Oxoacid Dehydrogenase Complex Lipoylation Correlation with Aerobiosis in Archaea. PLoS One, 9(1), e87063. doi:10.1371/journal.pone.0087063Bustamante-Marin, X. M., & Ostrowski, L. E. (2017). Cilia and Mucociliary Clearance. Cold Spring Harb Perspect Biol, 9(4). doi:10.1101/cshperspect.a028241Cao, X., Koch, T., Steffens, L., Finkensieper, J., Zigann, R., Cronan, J. E., & Dahl, C. (2018). Lipoate-binding proteins and specific lipoate-protein ligases in microbial sulfur oxidation reveal an atpyical role for an old cofactor. Elife, 7. doi:10.7554/eLife.37439The CCP4 suite: programs for protein crystallography. (1994). Acta Crystallogr D Biol Crystallogr, 50(Pt 5), 760-763. doi:10.1107/s0907444994003112Charlier, J., Barkema, H. W., Becher, P., De Benedictis, P., Hansson, I., Hennig- Pauka, I., . . . Zadoks, R. N. (2022). Disease control tools to secure animal and public health in a densely populated world. Lancet Planet Health, 6(10), e812-e824. doi:10.1016/s2542-5196(22)00147-4Chayen, N. E., & Saridakis, E. (2008). Protein crystallization: from purified protein to diffraction-quality crystal. Nature Methods, 5(2), 147-153. doi:10.1038/nmeth.f.203Christensen, Q. H., & Cronan, J. E. (2009). The Thermoplasma acidophilum LplA- LplB Complex Defines a New Class of Bipartite Lipoate-protein Ligases*.Journal of Biological Chemistry, 284(32), 21317-21326. doi:https://doi.org/10.1074/jbc.M109.015016Cianfrocco, M. A., Wong-Barnum, M., Youn, C., Wagner, R., & Leschziner, A.(2017). COSMIC2: A Science Gateway for Cryo-Electron Microscopy Structure Determination. Paper presented at the Proceedings of the Practice and Experience in Advanced Research Computing 2017 on Sustainability, Success and Impact, New Orleans, LA, USA. https://doi.org/10.1145/3093338.3093390Citti, C., Nouvel, L. X., & Baranowski, E. (2010). Phase and antigenic variation in mycoplasmas. Future Microbiol, 5(7), 1073-1085. doi:10.2217/fmb.10.71Cook, B. S., Beddow, J. G., Manso-Silván, L., Maglennon, G. A., & Rycroft, A. N. (2016). Selective medium for culture of Mycoplasma hyopneumoniae. Vet Microbiol, 195, 158-164. doi:10.1016/j.vetmic.2016.09.022Cronan, J. E. (2014). Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation. EcoSal Plus, 6(1). doi:10.1128/ecosalplus.ESP-0001-2012Cronan, J. E. (2016). Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway. Microbiol Mol Biol Rev, 80(2), 429-450. doi:10.1128/mmbr.00073-15Cronan, J. E. (2018). Advances in synthesis of biotin and assembly of lipoic acid. Curr Opin Chem Biol, 47, 60-66. doi:10.1016/j.cbpa.2018.08.004Crystal Structure Analysis: Principles and Practice. (2009). Oxford University Press.DeBey, M. C., & Ross, R. F. (1994). Ciliostasis and loss of cilia induced by Mycoplasma hyopneumoniae in porcine tracheal organ cultures. Infect Immun, 62(12), 5312-5318. doi:10.1128/iai.62.12.5312-5318.1994Douce, R., Bourguignon, J., Neuburger, M., & Rébeillé, F. (2001). The glycine decarboxylase system: a fascinating complex. Trends Plant Sci, 6(4), 167- 176. doi:10.1016/s1360-1385(01)01892-1Engel, N., van den Daele, K., Kolukisaoglu, U., Morgenthal, K., Weckwerth, W., Pärnik, T., . . . Bauwe, H. (2007). Deletion of glycine decarboxylase in Arabidopsis is lethal under nonphotorespiratory conditions. Plant Physiol, 144(3), 1328-1335. doi:10.1104/pp.107.099317Erdemir, D., Lee, A. Y., & Myerson, A. S. (2019). Crystal Nucleation. In A. Y. Lee, A. S. Myerson, & D. Erdemir (Eds.), Handbook of Industrial Crystallization (3 ed., pp. 76-114). Cambridge: Cambridge University Press.Ewald, R., Hoffmann, C., Florian, A., Neuhaus, E., Fernie, A. R., & Bauwe, H. (2014). Lipoate-Protein Ligase and Octanoyltransferase Are Essential for Protein Lipoylation in Mitochondria of Arabidopsis. Plant Physiol, 165(3), 978-990. doi:10.1104/pp.114.238311Fano, E., Pijoan, C., & Dee, S. (2005). Dynamics and persistence of Mycoplasma hyopneumoniae infection in pigs. Can J Vet Res, 69(3), 223-228.Fujiwara, K., Hosaka, H., Matsuda, M., Okamura-Ikeda, K., Motokawa, Y., Suzuki, M., . . . Taniguchi, H. (2007). Crystal Structure of Bovine Lipoyltransferase in Complex with Lipoyl-AMP. J Mol Biol, 371(1), 222-234. doi:https://doi.org/10.1016/j.jmb.2007.05.059 Fujiwara, K., Maita, N., Hosaka, H., Okamura-Ikeda, K., Nakagawa, A., &Taniguchi, H. (2010). Global conformational change associated with the two-step reaction catalyzed by Escherichia coli lipoate-protein ligase A. J Biol Chem, 285(13), 9971-9980. doi:10.1074/jbc.M109.078717Garg, A., & Gupta, D. (2008). VirulentPred: a SVM based prediction method for virulent proteins in bacterial pathogens. BMC Bioinformatics, 9(1), 62. doi:10.1186/1471-2105-9-62Gasteiger E., H. C., Gattiker A., Duvaud S., Wilkins M.R., Appel R.D., Bairoch A. (2005). Protein Identification and Analysis Tools on the Expasy Server. The Proteomics Protocols Handbook, pp. 571-560.Green, M. R., & Sambrook, J. (2020). The Inoue Method for Preparation and Transformation of Competent Escherichia coli: "Ultracompetent" Cells. Cold Spring Harb Protoc, 2020(6), 101196. doi:10.1101/pdb.prot101196Hansen, M. S., Pors, S. E., Jensen, H. E., Bille-Hansen, V., Bisgaard, M., Flachs, E. M., & Nielsen, O. L. (2010). An investigation of the pathology and pathogens associated with porcine respiratory disease complex in Denmark. J Comp Pathol, 143(2-3), 120-131. doi:10.1016/j.jcpa.2010.01.012Hao, Q. (2004). ABS: a program to determine absolute configuration and evaluate anomalous scatterer substructure. Journal of Applied Crystallography, 37(3), 498-499. doi:https://doi.org/10.1107/S0021889804008696Helke, K. L., Ezell, P. C., Duran-Struuck, R., & Swindle, M. M. (2015). Chapter 16 - Biology and Diseases of Swine. In J. G. Fox, L. C. Anderson, G. M. Otto, K. R. Pritchett-Corning, & M. T. Whary (Eds.), Laboratory Animal Medicine (Third Edition) (pp. 695-769). Boston: Academic Press.Himmelreich, R., Hilbert, H., Plagens, H., Pirkl, E., Li, B. C., & Herrmann, R. (1996). Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae. Nucleic Acids Res, 24(22), 4420-4449. doi:10.1093/nar/24.22.4420Jarocki, V. M., Raymond, B. B. A., Tacchi, J. L., Padula, M. P., & Djordjevic, S. P. (2019). Mycoplasma hyopneumoniae surface-associated proteases cleave bradykinin, substance P, neurokinin A and neuropeptide Y. Sci Rep, 9(1), 14585. doi:10.1038/s41598-019-51116-wJayson Beckman, F. G., Stephen Morgan, Ethan Sabala, Danielle J. Ufer, Adriana Valcu-Lisman, Wendy Zeng, and Shawn Arita. (2022). . Economic Research Report(ERR-310).Keeney, K. M., Stuckey, J. A., & O'Riordan, M. X. (2007). LplA1-dependent utilization of host lipoyl peptides enables Listeria cytosolic growth and virulence. Mol Microbiol, 66(3), 758-770. doi:10.1111/j.1365- 2958.2007.05956.xKim, D. J., Kim, K. H., Lee, H. H., Lee, S. J., Ha, J. Y., Yoon, H. J., & Suh, S. W. (2005). Crystal structure of lipoate-protein ligase A bound with the activated intermediate: insights into interaction with lipoyl domains. J Biol Chem, 280(45), 38081-38089. doi:10.1074/jbc.M507284200Kobisch, M., Quillien, L., Tillon, J. P., & Wróblewski, H. (1987). The Mycoplasmahyopneumoniae plasma membrane as a vaccine against porcine enzootic pneumonia. Ann Inst Pasteur Immunol, 138(5), 693-705. doi:10.1016/s0769-2625(87)80025-9Krissinel, E., & Henrick, K. (2007). Inference of macromolecular assemblies from crystalline state. J Mol Biol, 372(3), 774-797. doi:10.1016/j.jmb.2007.05.022Laskowski, R. A., & Swindells, M. B. (2011). LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model, 51(10), 2778- 2786. doi:10.1021/ci200227uLeal Zimmer, F. M. A., Paes, J. A., Zaha, A., & Ferreira, H. B. (2020). Pathogenicity & virulence of Mycoplasma hyopneumoniae. Virulence, 11(1), 1600-1622. doi:10.1080/21505594.2020.1842659Li, S., Fang, L., Liu, W., Song, T., Zhao, F., Zhang, R., . . . Xiao, S. (2019). Quantitative Proteomic Analyses of a Pathogenic Strain and Its Highly Passaged Attenuated Strain of Mycoplasma hyopneumoniae. Biomed Res Int, 2019, 4165735. doi:10.1155/2019/4165735Li, S., Fang, L., Liu, W., Song, T., Zhao, F., Zhang, R., . . . Xiao, S. (2019). Quantitative Proteomic Analyses of a Pathogenic Strain and Its Highly Passaged Attenuated Strain of Mycoplasma hyopneumoniae. Biomed Res Int, 2019, 4165735. doi:10.1155/2019/4165735Liu, W., Fang, L., Li, M., Li, S., Guo, S., Luo, R., . . . Xiao, S. (2012). Comparative genomics of Mycoplasma: analysis of conserved essential genes and diversity of the pan-genome. PLoS One, 7(4), e35698. doi:10.1371/journal.pone.0035698Liu, W., Xiao, S., Li, M., Guo, S., Li, S., Luo, R., . . . Fang, L. (2013). Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic 168 strain and its high-passaged attenuated strain. BMC Genomics, 14, 80. doi:10.1186/1471-2164-14-80Maes, D., Segales, J., Meyns, T., Sibila, M., Pieters, M., & Haesebrouck, F. (2008). Control of Mycoplasma hyopneumoniae infections in pigs. Vet Microbiol, 126(4), 297-309. doi:10.1016/j.vetmic.2007.09.008Maes, D., Sibila, M., Kuhnert, P., Segalés, J., Haesebrouck, F., & Pieters, M. (2018). Update on Mycoplasma hyopneumoniae infections in pigs: Knowledge gaps for improved disease control. Transbound Emerg Dis, 65 Suppl 1, 110-124. doi:10.1111/tbed.12677Maniloff, J. (1996). The minimal cell genome: "on being the right size". Proc Natl Acad Sci U S A, 93(19), 10004-10006. doi:10.1073/pnas.93.19.10004Marois-Créhan, C., Segalés, J., Holtkamp, D., Chae, C.-h., Deblanc, C., Opriessnig, T., & Fablet, C. (2021). Interactions of Mycoplasma hyopneumoniae with other pathogens and economic impact: CABI International.McPherson, A., & Gavira, J. A. (2014). Introduction to protein crystallization. Acta Crystallogr F Struct Biol Commun, 70(Pt 1), 2-20. doi:10.1107/s2053230x13033141Messerschmidt, & Albrecht. (2007). X-Ray Crystallography of Biomacromolecules:A Practical Guide: X-Ray Crystallography of Biomacromolecules: APractical Guide.Minion, F. C., Lefkowitz, E. J., Madsen, M. L., Cleary, B. J., Swartzell, S. M., &Mahairas, G. G. (2004). The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis. J Bacteriol, 186(21), 7123-7133. doi:10.1128/jb.186.21.7123-7133.2004Mistry, J., Chuguransky, S., Williams, L., Qureshi, M., Salazar, Gustavo A., Sonnhammer, E. L. L., . . . Bateman, A. (2020). Pfam: The protein families database in 2021. Nucleic Acids Res, 49(D1), D412-D419. doi:10.1093/nar/gkaa913 %J Nucleic Acids ResearchMorris, T. W., Reed, K. E., & Cronan, J. E., Jr. (1994). Identification of the gene encoding lipoate-protein ligase A of Escherichia coli. Molecular cloning and characterization of the lplA gene and gene product. J Biol Chem, 269(23), 16091-16100.O'Riordan, M., Moors, M. A., & Portnoy, D. A. (2003). Listeria intracellular growth and virulence require host-derived lipoic acid. Science, 302(5644), 462-464. doi:10.1126/science.1088170Opriessnig, T., Giménez-Lirola, L. G., & Halbur, P. G. (2011). Polymicrobial respiratory disease in pigs. Anim Health Res Rev, 12(2), 133-148. doi:10.1017/s1466252311000120Paes, J. A., Machado, L., Dos Anjos Leal, F. M., De Moraes, S. N., Moura, H., Barr, J. R., & Ferreira, H. B. (2018). Comparative proteomics of two Mycoplasma hyopneumoniae strains and Mycoplasma flocculare identified potential porcine enzootic pneumonia determinants. Virulence, 9(1), 1230-1246. doi:10.1080/21505594.2018.1499379Panjikar, S., Parthasarathy, V., Lamzin, V. S., Weiss, M. S., & Tucker, P. A. (2005). Auto-rickshaw: an automated crystal structure determination platform as an efficient tool for the validation of an X-ray diffraction experiment. Acta Crystallogr D Biol Crystallogr, 61(Pt 4), 449-457. doi:10.1107/s0907444905001307Pilatz, S., Breitbach, K., Hein, N., Fehlhaber, B., Schulze, J., Brenneke, B., . . . Steinmetz, I. (2006). Identification of Burkholderia pseudomallei genes required for the intracellular life cycle and in vivo virulence. Infect Immun, 74(6), 3576-3586. doi:10.1128/iai.01262-05Razin, S., Yogev, D., & Naot, Y. (1998). Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev, 62(4), 1094-1156. doi:10.1128/mmbr.62.4.1094-1156.1998Rei Yan, S. L., Wakasuqui, F., Du, X., Groves, M. R., & Wrenger, C. (2021). Lipoic Acid Metabolism as a Potential Chemotherapeutic Target Against Plasmodium falciparum and Staphylococcus aureus. Front Chem, 9, 742175. doi:10.3389/fchem.2021.742175Rowland, E. A., Snowden, C. K., & Cristea, I. M. (2018). Protein lipoylation: an evolutionarily conserved metabolic regulator of health and disease. CurrOpin Chem Biol, 42, 76-85. doi:10.1016/j.cbpa.2017.11.003Rucker, R. B., Morris, J., & Fascetti, A. J. (2008). Chapter 23 - Vitamins. In J. J. Kaneko, J. W. Harvey, & M. L. Bruss (Eds.), Clinical Biochemistry of Domestic Animals (Sixth Edition) (pp. 695-730). San Diego: Academic Press.Ruggeri, J., Salogni, C., Giovannini, S., Vitale, N., Boniotti, M. B., Corradi, A., . . .Alborali, G. L. (2020). Association Between Infectious Agents and Lesions in Post-Weaned Piglets and Fattening Heavy Pigs With Porcine Respiratory Disease Complex (PRDC). Front Vet Sci, 7, 636. doi:10.3389/fvets.2020.00636Schneider, T. R., & Sheldrick, G. M. (2002). Substructure solution with SHELXD. Acta Crystallogr D Biol Crystallogr, 58(Pt 10 Pt 2), 1772-1779. doi:10.1107/s0907444902011678Schonauer, M. S., Kastaniotis, A. J., Kursu, V. A., Hiltunen, J. K., & Dieckmann, C. L. (2009). Lipoic acid synthesis and attachment in yeast mitochondria. J Biol Chem, 284(35), 23234-23242. doi:10.1074/jbc.M109.015594Schrodinger, LLC. (2015). The PyMOL Molecular Graphics System, Version 1.8. Seymour, L. M., Deutscher, A. T., Jenkins, C., Kuit, T. A., Falconer, L., Minion, F. C., . . . Walker, M. J. (2010). A processed multidomain mycoplasma hyopneumoniae adhesin binds fibronectin, plasminogen, and swine respiratory cilia. J Biol Chem, 285(44), 33971-33978.doi:10.1074/jbc.M110.104463Shahbaaz, M., Bisetty, K., Ahmad, F., & Hassan, M. I. (2015). In silico approachesfor the identification of virulence candidates amongst hypothetical proteins of Mycoplasma pneumoniae 309. Comput Biol Chem, 59 Pt A, 67-80. doi:10.1016/j.compbiolchem.2015.09.007Shahbandeh, M. (2022). Projected pig meat consumption worldwide from 2021 to 2031. Statista, Consumer Goods & FMCG.Shahbandeh, M. (2023). Production of pork worldwide from 2013 to 2023. Statista, Consumer Goods & FMCG.Shapiro, S. (2013). Speculative strategies for new antibacterials: all roads should not lead to Rome. The Journal of Antibiotics, 66(7), 371-386. doi:10.1038/ja.2013.27Sheldrick, G. M. (2002). Macromolecular phasing with SHELXE. 217(12), 644- 650. doi:doi:10.1524/zkri.217.12.644.20662Sheldrick, G. M. (2010). Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr D Biol Crystallogr, 66(Pt 4), 479-485. doi:10.1107/s0907444909038360Sibila, M., Pieters, M., Molitor, T., Maes, D., Haesebrouck, F., & Segalés, J. (2009). Current perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection. Vet J, 181(3), 221-231. doi:10.1016/j.tvjl.2008.02.020Sibila, M., Pieters, M., Molitor, T., Maes, D., Haesebrouck, F., & Segalés, J. (2009). Current perspectives on the diagnosis and epidemiology of Mycoplasma hyopneumoniae infection. The Veterinary Journal, 181(3), 221-231. doi:https://doi.org/10.1016/j.tvjl.2008.02.020Simionatto, S., Marchioro, S. B., Maes, D., & Dellagostin, O. A. (2013). Mycoplasma hyopneumoniae: from disease to vaccine development. Vet Microbiol, 165(3-4), 234-242. doi:10.1016/j.vetmic.2013.04.019Siqueira, F. M., Thompson, C. E., Virginio, V. G., Gonchoroski, T., Reolon, L., Almeida, L. G., . . . Zaha, A. (2013). New insights on the biology of swine respiratory tract mycoplasmas from a comparative genome analysis. BMC Genomics, 14, 175. doi:10.1186/1471-2164-14-175Smyth, M. S., & Martin, J. H. (2000). x ray crystallography. Mol Pathol, 53(1), 8- 14. doi:10.1136/mp.53.1.8Solmonson, A., & DeBerardinis, R. J. (2018). Lipoic acid metabolism and mitochondrial redox regulation. J Biol Chem, 293(20), 7522-7530. doi:10.1074/jbc.TM117.000259Spalding, M. D., & Prigge, S. T. (2010). Lipoic acid metabolism in microbial pathogens. Microbiol Mol Biol Rev, 74(2), 200-228. doi:10.1128/mmbr.00008-10Tacchi, J. L., Raymond, B. B., Haynes, P. A., Berry, I. J., Widjaja, M., Bogema, D. R., . . . Djordjevic, S. P. (2016). Post-translational processing targets functionally diverse proteins in Mycoplasma hyopneumoniae. Open Biol, 6(2), 150210. doi:10.1098/rsob.150210Tajima, M., & Yagihashi, T. (1982). Interaction of Mycoplasma hyopneumoniae with the porcine respiratory epithelium as observed by electron microscopy. Infect Immun, 37(3), 1162-1169. doi:10.1128/iai.37.3.1162-1169.1982Taylor, G. L. (2010). Introduction to phasing. Acta Crystallogr D Biol Crystallogr, 66(Pt 4), 325-338. doi:10.1107/s0907444910006694Thacker, E. L. (2004). Diagnosis of Mycoplasma hyopneumoniae. Anim Health Res Rev, 5(2), 317-320. doi:10.1079/ahr200491Vangroenweghe, F., & Thas, O. (2021). Seasonal Variation in Prevalence of Mycoplasma hyopneumoniae and Other Respiratory Pathogens in Peri- Weaned, Post-Weaned, and Fattening Pigs with Clinical Signs of Respiratory Diseases in Belgian and Dutch Pig Herds, Using a Tracheobronchial Swab Sampling Technique, and Their Associations with Local Weather Conditions. Pathogens, 10(9). doi:10.3390/pathogens10091202Vasconcelos, A. T., Ferreira, H. B., Bizarro, C. V., Bonatto, S. L., Carvalho, M. O., Pinto, P. M., . . . Zaha, A. (2005). Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae. J Bacteriol, 187(16), 5568-5577. doi:10.1128/jb.187.16.5568-5577.2005Vekilov, P. G., & Vorontsova, M. A. (2014). Nucleation precursors in protein crystallization. Acta Crystallogr F Struct Biol Commun, 70(Pt 3), 271-282. doi:10.1107/s2053230x14002386Wang, G., Shen, Y., Li, C., Zhu, Q., & ZhanBota, A. (2022). The regulatory effect of herd structure on pig production under the environmental regulation. PLoS One, 17(4), e0266687. doi:10.1371/journal.pone.0266687Wang, Q.-S., Zhang, K.-H., Cui, Y., Wang, Z.-J., Pan, Q.-Y., Liu, K., . . . He, J.-H. (2018). Upgrade of macromolecular crystallography beamline BL17U1 at SSRF. Nuclear Science and Techniques, 29(5), 68. doi:10.1007/s41365- 018-0398-9Wang, X., Li, K., Qin, X., Li, M., Liu, Y., An, Y., . . . Gong, J. (2022). Research on Mesoscale Nucleation and Growth Processes in Solution Crystallization: A Review. 12(9), 1234. Retrieved from https://www.mdpi.com/2073- 4352/12/9/1234Westberg, J., Persson, A., Holmberg, A., Goesmann, A., Lundeberg, J., Johansson, K. E., . . . Uhlén, M. (2004). The genome sequence of Mycoplasma mycoides subsp. mycoides SC type strain PG1T, the causative agent of contagious bovine pleuropneumonia (CBPP). Genome Res, 14(2), 221-227. doi:10.1101/gr.1673304Wilkins, M. R., Gasteiger, E., Bairoch, A., Sanchez, J. C., Williams, K. L., Appel, R. D., & Hochstrasser, D. F. (1999). Protein identification and analysis tools in the ExPASy server. Methods Mol Biol, 112, 531-552. doi:10.1385/1- 59259-584-7:531Yang, X., Yin, N., Pang, D., Wu, K., Yin, Y., & Zhang, X. (2010). [Contributions of putative lipoate-protein ligase to the virulence of Streptococcus pneumoniae]. Wei Sheng Wu Xue Bao, 50(6), 774-779.Zhang, B., Ku, X., Yu, X., Sun, Q., Wu, H., Chen, F., . . . He, Q. (2019). Prevalence and antimicrobial susceptibilities of bacterial pathogens in Chinese pig farms from 2013 to 2017. Sci Rep, 9(1), 9908. doi:10.1038/s41598-019- 45482-8Zhang, X., Nie, J., Zheng, Y., Ren, J., & Zeng, A. P. (2020). Activation and competition of lipoylation of H protein and its hydrolysis in a reaction cascade catalyzed by the multifunctional enzyme lipoate-protein ligase A. Biotechnol Bioeng, 117(12), 3677-3687. doi:10.1002/bit.27526Zhu, K., Chen, H., Jin, J., Wang, N., Ma, G., Huang, J., . . . Liu, H. (2020). Functional Identification and Structural Analysis of a New Lipoate Protein Ligase in Mycoplasma hyopneumoniae. Front Cell Infect Microbiol, 10, 156. doi:10.3389/fcimb.2020.00156