Fumarase C (FumC) catalyzes the reversible conversion of fumarate to S‐malate. Previous structural investigations within the superfamily have reported a dynamic structural segment, termed the SS Loop. To date, active‐site asymmetry has raised the question of how SS Loop placement affects participation of key residues during the reaction. Herein, we report structural and kinetic analyses from Escherichia coli FumC variants to understand the contribution of SS Loop residues S318, K324, and N326. High‐resolution X‐ray crystallographic results reveal three distinct FumC active‐site conformations; disordered‐open, ordered‐open, and the newly discovered ordered‐closed. Surprisingly, each SS Loop variant has unaffected Michaelis constants coupled to reductions in turnover number. Based upon our structural and functional analyses, we propose structural and catalytic roles for each of the aforementioned residues.
As bacterial pathogens gain antibacterial resistance, few mechanisms remain available for halting pathogenicity, giving a frightening glimpse into a post‐antibacterial era. However, the secretion of virulence factors is often imperative to bacterial pathogenesis within the human body and is becoming a larger target for antibiotic treatment. The two‐partner secretion (TPS) pathway, harboring both A (TpsA) and B‐components (TpsB), is the most commonly used gram‐negative virulence factor secretion system currently known. In fact, whooping cough, meningitis, UTIs, and certain food‐borne illnesses have been attributed to gram‐negative bacterial species containing the TPS system. Systematically, TpsA members are activated concomitant with TpsB‐dependent secretion across the outer membrane. Upon secretion, TpsA members elicit a variety of functions including cytolysis, adhesion, contact‐growth inhibition, and iron sequestration thus allowing the pathogen to invade and proliferate within the host, advantageously. Structurally, TpsA members can be divided into a TPS domain and a functional (virulent) domain. All TPS domains are constructed from a ~300‐residue right‐handed, parallel, β‐helix, and recognize their cognate TpsB membrane‐bound partner during secretion. Fundamentally, TpsA β‐helix structures are built from consecutive β‐circuits, where each β‐circuit is constructed from three parallel β‐strands. In order to further understand the relationship between complete TpsA β‐circuit establishment, structural stability and function we have implemented a truncated form of hemolysin A (HpmA265) from Proteus mirabilis. In previous studies, HpmA265 was structurally separated into three sequential folding subdomains: polar core, non‐polar core, and carboxy‐terminus. Structurally, the carboxy‐terminal subdomain harbors a partial, two‐stranded β‐circuit. Previous investigations replaced valine 158 (V158) and phenylalanine 215 (F215), as located within the first and last parallel β‐strands of the non‐polar core subdomain, with polar residues. The site‐selective alteration of V158 demonstrated increased function, while merging the unfolding transitions associated with the non‐polar and carboxy‐terminal subdomains. Alternatively, site‐selective alteration of F215 demonstrated decreased function, while destabilizing the transitions associated with both the non‐polar and carboxy‐terminal subdomains. Ultimately, template‐assisted activity has been interrelated to extent of β‐circuit structural destabilization within the carboxy‐terminal subdomain. Specifically, this research expands upon the previous V158 and F215 results by probing the structural and functional relationship via progressive amino‐acid extension to the HpmA265 carboxy‐terminal subdomain. Ultimately, the structural and functional effects of final β‐circuit completion will be ascertained using protein unfolding and hemolytic functional measurements.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The secretion of virulence factors often aids bacterial pathogenesis. The two‐partner secretion (TPS) pathway, harboring both A (TpsA) and B‐components (TpsB), is the most commonly used gram‐negative virulence factor secretion system. In fact, whooping cough, meningitis, and certain food‐borne illnesses have been attributed to TPS pathway containing gram‐negative bacterial species. Systematically, TpsA members are activated concomitant with Tps‐dependent secretion across the outer membrane. Upon secretion, TpsA members elicit a variety of functions including cytolysis, adhesion, contact‐growth inhibition, and iron sequestration. Collectively, these TpsA functions provide advantageous invasion and proliferation strategies within host cells. Structurally, TpsA members can be divided into a two‐partner secretion (TPS) domain and a functional domain. All TPS domains are constructed from a 300‐residue right‐handed, parallel β‐helix structure, and recognize their cognate TpsB partner. In order to further understand the relationship between TPS domains and TpsA structure and function, we have implemented a truncated form of hemolysin A (HpmA265), a TpsA member from Proteus mirabilis. In previous studies, HpmA265 was structurally separated into three sequential folding subdomains: polar core, non‐polar core, and carboxy‐terminus. Specifically, these research investigations targeted valine 158 (V158) and phenylalanine 215 (F215) located within the first and last parallel β‐strands of the non‐polar core subdomain. A series of site‐selective variations were established at both V158 and F215. These variant forms of HpmA265 were characterized structurally via protease sensitivity and protein folding techniques, while functionality was ascertained within a template‐assisted hemolysis assay. Structurally, the V158 variants have destabilized the unfolding transitions associated with both the polar and non‐polar core subdomains, while leaving functionality unaffected. Site‐selective variants at F215 have selectively destabilized the non‐polar core subdomain, while leaving the unfolding transition attributed to the polar core subdomain unaffected. Additionally, the F215 variants do not affect template‐assisted hemolysis. Therefore, our results have been able to dissect the structural stability within the non‐polar core subdomain from template‐assisted function. These results have expanded the understanding for the implementation of TPS domains within gram‐negative bacteria.Support or Funding InformationFunding for this research was provided by: University Wisconsin – La Crosse Faculty Research Grant Program (TMW) and University Wisconsin – La Crosse Undergraduate Research and Creativity Grant Program (JDG).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.