Selectivity within a Family of Bacterial Phosphothreonine Lyases

Abstract

Phosphothreonine lyases are bacterial effector proteins secreted into host cells to facilitate the infection process. This enzyme family catalyzes an irreversible elimination reaction that converts phosphothreonine or phosphoserine to dehydrobutyrine or dehydroalanine, respectively. Herein, we report a study of substrate selectivity for each of the four known phosphothreonine lyases. This was accomplished using a combination of mass spectrometry and enzyme kinetics assays for a series of phosphorylated peptides derived from the mitogen-activated protein kinase (MAPK) activation loop. These studies provide the first experimental evidence that VirA, a putative phosphothreonine lyase identified through homology, is indeed capable of catalyzing phosphate elimination. These studies further demonstrate that OspF is the most promiscuous phosphothreonine lyase, whereas SpvC is the most specific for the MAPK activation loop. Our studies reveal that phospholyases are dramatically more efficient at catalyzing elimination from phosphothreonine than from phosphoserine. Together, our data suggest that each enzyme likely has preferred substrates, either within the MAPK family or beyond. Fully understanding the extent of selectivity is key to understanding the impact of phosphothreonine lyases during bacterial infection and to exploiting their unique chemistry for a range of applications.

LanCLs add glutathione to dehydroamino acids generated at phosphorylated sites in the proteome

“…The bacterial pathogen pThr lyases SpvC and OspF catalyze phosphate elimination from mitogen activated protein kinase (MAPK) activation loop peptides containing pThr to form the corresponding Dhb-containing peptides (Chambers et al, 2018;Zhu et al, 2007). We therefore used these lyases to mimic such pathogen-induced damage to prepare model, Dhb-containing peptides corresponding to the activation loops of ERK and Akt (Figure 2A; Figure S1C).…”

A Chemical Probe for Dehydrobutyrine

“…Currently, the only confirmed mammalian targets of bacterial phospholyases are MAP kinases, including ERK1/2. Our prior work implied that phospholyases may have additional targets outside of the MAPK family . Moreover, this work has serendipitously identified histone H3 as a previously undiscovered target of OspF (Figure S7).…”

“…Previous work has established that, during bacterial infection, phospholyases attenuate the innate immune response by deactivating mitogen‐activated protein kinase (MAPK) signaling through β‐elimination of phosphate from a key pThr within the ERK1/2 activation loop –. Our recent work has demonstrated that phospholyases may have additional targets beyond the MAPK family . Identification of new phospholyase targets would lead to a better understanding of the infection process, and could uncover valuable biomarkers for bacterial infection.…”

Dehydroamino acid chemical biology: an example of functional group interconversion on proteins

“… 10 Using an in vitro LC-MS assay that measured the conversion of p-Thr and p-Ser containing peptides to the respective dehydroamino acids, it was possible to assess the substrate specificity of several bacterial phospholyases. 11 This work suggested that the promiscuous phospholyase activity of OspF may be responsible for the increased pathogenicity of Shigella compared to other bacteria such as Salmonella .…”