Biophysical and Biochemical Characterization of TP0037, a d -Lactate Dehydrogenase, Supports an Acetogenic Energy Conservation Pathway in Treponema pallidum

Abstract

A longstanding conundrum in Treponema pallidum biology concerns how the spirochete generates sufficient energy to fulfill its complex pathogenesis processes during human syphilitic infection. For decades, it has been assumed that the bacterium relies solely on glucose catabolism (via glycolysis) for generation of its ATP. However, the organism’s robust motility, believed to be essential for human tissue invasion and dissemination, would require abundant ATP likely not provided by the parsimony of glycolysis. As such, additional ATP generation, either via a chemiosmotic gradient, substrate-level phosphorylation, or both, likely exists in T. pallidum. Along these lines, we have hypothesized that T. pallidum exploits an acetogenic energy conservation pathway that relies on the redox chemistry of flavins. Central to this hypothesis is the apparent existence in T. pallidum of an acetogenic pathway for the conversion of d-lactate to acetate. Herein we have characterized the structural, biophysical, and biochemical properties of the first enzyme (d-lactate dehydrogenase [d-LDH]; TP0037) predicted in this pathway. Binding and enzymatic studies showed that recombinant TP0037 consumed d-lactate and NAD+ to produce pyruvate and NADH. The crystal structure of TP0037 revealed a fold similar to that of other d-acid dehydrogenases; residues in the cofactor-binding and active sites were homologous to those of other known d-LDHs. The crystal structure and solution biophysical experiments revealed the protein’s propensity to dimerize, akin to other d-LDHs. This study is the first to elucidate the enzymatic properties of T. pallidum’s d-LDH, thereby providing new compelling evidence for a flavin-dependent acetogenic energy conservation (ATP-generating) pathway in T. pallidum. IMPORTANCE Because T. pallidum lacks a Krebs cycle and the capability for oxidative phosphorylation, historically it has been difficult to reconcile how the syphilis spirochete generates sufficient ATP to fulfill its energy needs, particularly for its robust motility, solely from glycolysis. We have postulated the existence in T. pallidum of a flavin-dependent acetogenic energy conservation pathway that would generate additional ATP for T. pallidum bioenergetics. In the proposed acetogenic pathway, first d-lactate would be converted to pyruvate. Pyruvate would then be metabolized to acetate in three additional steps, with ATP being generated via substrate-level phosphorylation. This study provides structural, biochemical, and biophysical evidence for the first T. pallidum enzyme in the pathway (TP0037; d-lactate dehydrogenase) requisite for the conversion of d-lactate to pyruvate. The findings represent the first experimental evidence to support a role for an acetogenic energy conservation pathway that would contribute to nonglycolytic ATP production in T. pallidum.

Biophysical and biochemical studies support TP0094 as a phosphotransacetylase in an acetogenic energy-conservation pathway in Treponema pallidum

“…pallidum , we have confirmed the activity of only one of them: the D-lactate dehydrogenase encoded by gene tp0037 (TpDLD; 1.1.1.28; UniProt Acc. # O83080) [ 11 ].…”

Inhibition of bacterial FMN transferase: A potential avenue for countering antimicrobial resistance

“…FMNylation potentially is a vulnerable "Achilles heel" in pathogenic bacteria because this reaction is necessary for the proper formation of flavin-and quinonebased redox-driven Na + pumps (NQR, Na + -translocating NADH:quinone oxidoreductase and RNF, Rhodobacter nitrogen fixation) that often provide the only ion-motive (chemiosmotic gradient) electron-transport chain in these organisms. [7][8][9][10][11] Additionally, Ftp homologs are not present in higher eukaryotes. 12 It is now appreciated that Ftp's FMN-transferase activity is required for the periplasmic FMNylation and electron-transfer functions of subunits (e.g., NqrC and RnfG) of these flavin-based cytoplasmic membrane-bound NQR and/or RNF redox complexes, providing a link between those periplasmic functions and the redox reactions that occur within the bacterial cytoplasm.…”

“…1,[13][14][15] Disruption of the chemiosmotic gradient generated by these redox complexes has the potential to arrest essential bacterial functions such as ATP synthesis, flagellar rotation, and nutrient transport. 11,16,17 Indeed, interruption of the Ftp gene in Listeria monocytogenes limited the bacterium's ability to grow on a carbon source that requires extracellular electron transport for utilization. 18 Thus, inhibitors of FMNylation (i.e., of Ftp) have the potential to curtail bacterial growth and therefore could lead to new antimicrobials to combat antibiotic-resistant bacteria, which are a growing menace.…”

“…FMNylation potentially is a vulnerable “Achilles heel” in pathogenic bacteria because this reaction is necessary for the proper formation of flavin‐ and quinone‐based redox‐driven Na + pumps (NQR, Na + ‐translocating NADH:quinone oxidoreductase and RNF, Rhodobacter nitrogen fixation) that often provide the only ion‐motive (chemiosmotic gradient) electron‐transport chain in these organisms 7–11 . Additionally, Ftp homologs are not present in higher eukaryotes 12 .…”

“…It is now appreciated that Ftp's FMN‐transferase activity is required for the periplasmic FMNylation and electron‐transfer functions of subunits (e.g., NqrC and RnfG) of these flavin‐based cytoplasmic membrane‐bound NQR and/or RNF redox complexes, providing a link between those periplasmic functions and the redox reactions that occur within the bacterial cytoplasm 1,13–15 . Disruption of the chemiosmotic gradient generated by these redox complexes has the potential to arrest essential bacterial functions such as ATP synthesis, flagellar rotation, and nutrient transport 11,16,17 . Indeed, interruption of the Ftp gene in Listeria monocytogenes limited the bacterium's ability to grow on a carbon source that requires extracellular electron transport for utilization 18 .…”