A Conserved Domain in
            <i>Escherichia coli</i>
            Lon Protease Is Involved in Substrate Discriminator Activity

Ebel, Wolfgang; Skinner, Monica M.; Dierksen, Karen P.; Scott, Janelle M.; Trempy, Janine E.

doi:10.1128/jb.181.7.2236-2243.1999

Cited by 57 publications

(26 citation statements)

References 62 publications

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“…2). The phenomenon of highly variable N-terminal and SSD domains is in agreement with the finding that they are responsible for the discriminatory recognition of specific substrates [34,48].…”

Section: Discussionsupporting

confidence: 89%

Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR‐249 and biochemical characterization of its thermostable recombinant enzyme

Tsay

Chen

2004

European Journal of Biochemistry

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A gene encoding thermostable Lon protease from Brevibacillus thermoruber WR-249 was cloned and characterized. The Br. thermoruber Lon gene (Bt-lon) encodes an 88 kDa protein characterized by an N-terminal domain, a central ATPase domain which includes an SSD (sensor-and substrate-discrimination) domain, and a C-terminal protease domain. The Bt-lon is a heat-inducible gene and may be controlled under a putative Bacillus subtilis r A -dependent promoter, but in the absence of CIRCE (controlling inverted repeat of chaperone expression). Bt-lon was expressed in Escherichia coli, and its protein product was purified. The native recombinant Br. thermoruber Lon protease (Bt-Lon) displayed a hexameric structure. The optimal temperature of ATPase activity for Bt-Lon was 70°C, and the optimal temperature of peptidase and DNA-binding activities was 50°C. This implies that the functions of Lon protease in thermophilic bacteria may be switched, depending on temperature, to regulate their physiological needs. The peptidase activity of Bt-Lon increases substantially in the presence of ATP. Furthermore, the substrate specificity of Bt-Lon is different from that of E. coli Lon in using fluorogenic peptides as substrates. Notably, the Bt-Lon protein shows chaperone-like activity by preventing aggregation of denatured insulin B-chain in a dose-dependent and ATPindependent manner. In thermal denaturation experiments, Bt-Lon was found to display an indicator of thermostability value, T m of 71.5°C. Sequence comparison with mesophilic Lon proteases shows differences in the rigidity, electrostatic interactions, and hydrogen bonding of Bt-Lon relevant to thermostability.

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Section: Discussionsupporting

confidence: 89%

Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR‐249 and biochemical characterization of its thermostable recombinant enzyme

Tsay

Chen

2004

European Journal of Biochemistry

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“…We hypothesized that the altered Congo red staining of the lon-defective (lon::FRT) strain, compared to the WT strain, might be due to an altered cell envelope. In E. coli, Lon protease affects capsule production through degradation of an activator for capsular polysaccharide biosynthesis (40). To determine whether surA1 had a direct role in the observed phenotypes, we constructed a double deletion mutant (lon::FRT and surA1::Km).…”

Section: Resultsmentioning

confidence: 99%

Lon Protease Has Multifaceted Biological Functions in Acinetobacter baumannii

et al. 2019

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Acinetobacter baumannii is a Gram-negative opportunistic pathogen that is known to survive harsh environmental conditions and is a leading cause of hospital-acquired infections. Specifically, multicellular communities (known as biofilms) of A. baumannii can withstand desiccation and survive on hospital surfaces and equipment. Biofilms are bacteria embedded in a self-produced extracellular matrix composed of proteins, sugars, and/or DNA. Bacteria in a biofilm are protected from environmental stresses, including antibiotics, which provides the bacteria with selective advantage for survival. Although some gene products are known to play roles in this developmental process in A. baumannii, mechanisms and signaling remain mostly unknown. Here, we find that Lon protease in A. baumannii affects biofilm development and has other important physiological roles, including motility and the cell envelope. Lon proteases are found in all domains of life, participating in regulatory processes and maintaining cellular homeostasis. These data reveal the importance of Lon protease in influencing key A. baumannii processes to survive stress and to maintain viability. IMPORTANCE Acinetobacter baumannii is an opportunistic pathogen and is a leading cause of hospital-acquired infections. A. baumannii is difficult to eradicate and to manage, because this bacterium is known to robustly survive desiccation and to quickly gain antibiotic resistance. We sought to investigate biofilm formation in A. baumannii, since much remains unknown about biofilm formation in this bacterium. Biofilms, which are multicellular communities of bacteria, are surface attached and difficult to eliminate from hospital equipment and implanted devices. Our research identifies multifaceted physiological roles for the conserved bacterial protease Lon in A. baumannii. These roles include biofilm formation, motility, and viability. This work broadly affects and expands understanding of the biology of A. baumannii, which will permit us to find effective ways to eliminate the bacterium.

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“…In addition to its AAA+ module and peptidase domain, E. coli Lon contains a family‐specific N domain that is necessary but not sufficient for hexamerization and has been proposed to be involved in substrate binding (Roudiak and Shrader, ; Ebel et al ., ; Rudyak and Shrader, ; Melnikov et al ., ; Adam et al ., ). Consistently, N‐domain mutations or truncations result in defects in Lon activity in vitro (Cheng et al ., ), but substrate binding to the N domain has not been directly demonstrated.…”

Section: Introductionmentioning

confidence: 99%

Roles of the N domain of the AAA+ Lon protease in substrate recognition, allosteric regulation and chaperone activity

Wohlever

Baker

Sauer

2013

Molecular Microbiology

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Summary Degron binding regulates the activities of the AAA+ Lon protease in addition to targeting proteins for degradation. The sul20 degron from the cell-division inhibitor SulA is shown here to bind to the N domain of E. coli Lon, and the recognition site is identified by crosslinking and scanning for mutations that prevent sul20-peptide binding. These N-domain mutations limit the rates of proteolysis of model sul20-tagged substrates and ATP hydrolysis by an allosteric mechanism. Lon inactivation of SulA in vivo requires binding to the N domain and robust ATP hydrolysis but does not require degradation or translocation into the proteolytic chamber. Lon-mediated relief of proteotoxic stress and protein aggregation in vivo can also occur without degradation but is not dependent on robust ATP hydrolysis. In combination, these results demonstrate that Lon can function as a protease or a chaperone and reveal that some of its ATP-dependent biological activities do not require translocation.

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A Conserved Domain in Escherichia coli Lon Protease Is Involved in Substrate Discriminator Activity

Cited by 57 publications

References 62 publications

Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR‐249 and biochemical characterization of its thermostable recombinant enzyme

Identification of a gene encoding Lon protease from Brevibacillus thermoruber WR‐249 and biochemical characterization of its thermostable recombinant enzyme

Lon Protease Has Multifaceted Biological Functions in Acinetobacter baumannii

Roles of the N domain of the AAA+ Lon protease in substrate recognition, allosteric regulation and chaperone activity

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