Functional Identification and Structural Analysis of a New Lipoate Protein Ligase in Mycoplasma hyopneumoniae

Abstract

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the causative agent of pandemic pneumonia among pigs, namely, swine enzootic pneumonia. Although M. hyopneumoniae was first identified in 1965, little is known regarding its metabolic pathways, which might play a pivotal role during disease pathogenesis. Lipoate is an essential cofactor for enzymes important for central metabolism. However, the lipoate metabolism pathway in M. hyopneumoniae is definitely unclear. Here, we identified a novel gene, lpl, encoding a lipoate protein ligase in the genome of M. hyopneumoniae (Mhp-Lpl). This gene contains 1,032 base pairs and encodes a protein of 343 amino acids, which is between 7.5 and 36.09% identical to lipoate protein ligases (Lpls) of other species. Similar to its homologs in other species, Mhp-Lpl catalyzes the ATP-dependent activation of lipoate to lipoyl-AMP and the transfer of the activated lipoyl onto the lipoyl domains of M. hyopneumoniae GcvH (Mhp H) in vitro. Enzymatic and mutagenesis analysis indicate that residue K56 within the SKT sequence of Mhp H protein is the lipoyl moiety acceptor site. The three-dimensional structure showed typical lipoate protein ligase folding, with a large N-terminal domain and a small C-terminal domain. The large N-terminal domain is responsible for the full enzymatic activity of Mhp-Lpl. The identification and characterization of Mhp-Lpl will be beneficial to our understanding of M. hyopneumoniae metabolism.

A Novel Lipoate-Protein Ligase, Mhp-LplJ, Is Required for Lipoic Acid Metabolism in Mycoplasma hyopneumoniae

“…A New Putative Lipoate-Protein Ligase Exists in M. hyopneumoniae Previously, we reported that Mhp-Lpl catalyzes the attachment of lipoic acid to the lipoyl domains of GcvH in vitro (Zhu et al, 2020). We wondered whether the lipoate modification of PdhD is catalyzed by Mhp-Lpl.…”

“…Lipoate-protein ligases are responsible for the lipoate modification of these enzymes ( Reed et al, 1958 ). We previously reported that Mhp-Lpl of M. hyopneumoniae is a member of the lipoate-protein ligase family and can catalyze the lipoylation of M. hyopneumoniae GcvH in vitro ( Zhu et al, 2020 ). However, unexpectedly, Mhp-Lpl did not lipoylate PdhD, a lipoate-dependent subunit of M. hyopneumoniae ( Figure 3A ).…”

“…To date, two lipoate-protein ligases, Mhp-Lpl ( Zhu et al, 2020 ) and Mhp-LplJ, have been identified in M. hyopneumoniae. It is uncommon for bacteria to have two lipoate-protein ligases responsible for the salvage pathway of lipoic acid metabolism.…”

“…In mycoplasma species, lipoic acid metabolism is poorly understood. We previously reported a lipoate-protein ligase, Mhp-Lpl, in M. hyopneumoniae ( Zhu et al, 2020 ). However, we found Mhp-Lpl is not able to modify dihydrolipoamide dehydrogenase (PdhD), a lipoate-requiring protein of M. hyopneumoniae .…”

Infection strategies of mycoplasmas: Unraveling the panoply of virulence factors

“…Lipoate protein ligase A (lplA) plays a role in the ligation of lipoic acid from host cells to the E2 subunit of the PDH complex to generate E2-lipoamide, which ultimately plays a crucial role in pyruvate metabolism [255]. lplA has been shown to be upregulated in the pathogenic 168 strain of M. hyopneumoniae [256].…”