Structural insights into Mycoplasma hyopneumoniae (Mhp) lipoate protein ligase A (LplA)

Abstract

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.