An Essential GTPase, Der, Containing Double GTP-binding Domains from Escherichia coli and Thermotoga maritima

Hwang, Jihwan; Inouye, Masayori

doi:10.1074/jbc.m104455200

Cited by 62 publications

(96 citation statements)

References 20 publications

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“…For the expression of TmDer and EcDer, pETTmDer 8,13 or pETEcDer 12 was introduced into BL21(DE3). The expression and purification of TmDer was carried out as described previously.…”

Section: Purification Of Gtpase Proteinmentioning

confidence: 99%

“…The expression and purification of TmDer was carried out as described previously. 8 EcDer was overexpressed and purified with a similar protocol except for a heat treatment. The open reading frames of der in Deinococcus radiodurans and Staphylococcus aureus were cloned in NdeI-EcoRI sites of pET28a (N-terminal His tag fusion, Novagen, Gibbstown, NJ, USA), yielding pET28DrDer and pET28SaDer.…”

Section: Purification Of Gtpase Proteinmentioning

confidence: 99%

“…[4][5][6] Even though there is low primary sequence similarity among different GTPase families, most GTPases have an impressively common structural topology consisting of six b-strands surrounded by five a-helices. 4,5,7 Previously we characterized an essential GTPase, Der (double Eralike protein), from Escherichia coli (EcDer) [8][9][10][11][12] and from a hyperthermophilic bacterium Thermotoga maritima (TmDer). 8,13 The 3D structure of TmDer was solved by X-ray crystallography.…”

Section: Introductionmentioning

confidence: 99%

“…4,5,7 Previously we characterized an essential GTPase, Der (double Eralike protein), from Escherichia coli (EcDer) [8][9][10][11][12] and from a hyperthermophilic bacterium Thermotoga maritima (TmDer). 8,13 The 3D structure of TmDer was solved by X-ray crystallography. 13 Der homologs among other eubacteria, including E. coli, show highly conserved sequence homology from the N-terminus to the C-terminus.…”

Section: Introductionmentioning

confidence: 99%

“…13 Der homologs among other eubacteria, including E. coli, show highly conserved sequence homology from the N-terminus to the C-terminus. 8 Der has a C-terminal KH-like (RNA-binding module-like) domain flanked by two tandemly repeated GTP-binding domains at the N-terminus. 13,14 In case of T. maritima Der, each of the two Der GTP-binding domains expressed separately has an intrinsic GTPase activity, though the activity of the N-terminal GD1 domain is stronger than that of the GD2 domain.…”

Section: Introductionmentioning

confidence: 99%

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Structure-based design and screening of inhibitors for an essential bacterial GTPase, Der

Hwang

Tseitin²,

Ramnarayan³

et al. 2012

J Antibiot

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Der is an essential and widely conserved GTPase that assists assembly of a large ribosomal subunit in bacteria. Der associates specifically with the 50S subunit in a GTP-dependent manner and the cells depleted of Der accumulate the structurally unstable 50S subunit, which dissociates into an aberrant subunit at a lower Mg 2+ concentration. As Der is an essential and ubiquitous protein in bacteria, it may prove to be an ideal cellular target against which new antibiotics can be developed. In the present study, we describe our attempts to identify novel antibiotics specifically targeting Der GTPase. We performed the structure-based design of Der inhibitors using the X-ray crystal structure of Thermotoga maritima Der (TmDer). Virtual screening of commercially available chemical library retrieved 257 small molecules that potentially inhibit Der GTPase activity. These 257 chemicals were tested for their in vitro effects on TmDer GTPase and in vivo antibacterial activities. We identified three structurally diverse compounds, SBI-34462, -34566 and -34612, that are both biologically active against bacterial cells and putative enzymatic inhibitors of Der GTPase homologs. We also presented the possible interactions of each compound with the Der GTP-binding site to understand the mechanism of inhibition. Therefore, our lead compounds inhibiting Der GTPase provide scaffolds for the development of novel antibiotics against antibiotic-resistant pathogenic bacteria.

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“…For the expression of TmDer and EcDer, pETTmDer 8,13 or pETEcDer 12 was introduced into BL21(DE3). The expression and purification of TmDer was carried out as described previously.…”

Section: Purification Of Gtpase Proteinmentioning

confidence: 99%

Section: Purification Of Gtpase Proteinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Structure-based design and screening of inhibitors for an essential bacterial GTPase, Der

Hwang

Tseitin²,

Ramnarayan³

et al. 2012

J Antibiot

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Analysis of GTPases carrying hydrophobic amino acid substitutions in lieu of the catalytic glutamine: Implications for GTP hydrolysis

et al. 2005

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Ras superfamily GTP-binding proteins regulate important signaling events in the cell. Ras, which often serves as a prototype, efficiently hydrolyzes GTP in conjunction with its regulator GAP. A conserved glutamine plays a vital role in GTP hydrolysis in most GTP-binding proteins. Mutating this glutamine in Ras has oncogenic effects, since it disrupts GTP hydrolysis. The analysis presented here is of GTP-binding proteins that are a paradox to oncogenic Ras, since they have the catalytic glutamine (Glncat) substituted by a hydrophobic amino acid, yet can hydrolyze GTP efficiently. We term these proteins HAS-GTPases. Analysis of the amino acid sequences of HAS-GTPases reveals prominent presence of insertions around the GTP-binding pocket. Homology modeling studies suggest an interesting means to achieve catalysis despite the drastic hydrophobic substitution replacing the key Glncat of Ras-like GTPases. The substituted hydrophobic residue adopts a "retracted conformation," where it is positioned away from the GTP, as its role in catalysis would be unproductive. This conformation is further stabilized by interactions with hydrophobic residues in its vicinity. These interacting residues are strongly conserved and hydrophobic in all HAS-GTPases, and correspond to residues Asp92 and Tyr96 of Ras. An experimental support for the "retracted conformation" of Switch II arises from the crystal structures of Ylqf and hGBP1. This conformation allows us to hypothesize that, unlike in classical GTPases, catalytic residues could be supplied by regions other than the Switch II (i.e., either the insertions or a neighboring domain).

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Expression of Vitreoscilla hemoglobin enhances growth and levels of α-amylase in Schwanniomyces occidentalis

Suthar

Chattoo

2006

Appl Microbiol Biotechnol

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A metabolic engineering approach was exploited to improve growth and protein secretion in the non-conventional yeast, Schwanniomyces occidentalis. Vitreoscilla hemoglobin (VHb) gene was expressed in S. occidentalis under the control of the native alpha-amylase (AMY1) promoter. Expression of VHb was confirmed by reverse transcriptase polymerase chain reaction and Western blot hybridization analysis. Effect of VHb on growth and protein secretion was studied in synthetic medium under both limiting and non-limiting dissolved oxygen conditions. Under both conditions, VHb-expressing cells exhibited higher oxygen uptake and higher specific growth rates. Levels of extracellular alpha-amylase were also elevated in the VHb-transformed strain relative to the control strain. In amylase production medium, VHb-expressing cells showed 3-fold elevated levels of alpha-amylase and a 31% increase in the total secreted protein under oxygen-limiting environment. VHb was found to localize in the mitochondria in addition to its cytoplasmic location. Inhibition of respiration by antimycin A resulted in the loss of the growth-enhancing effects of VHb. A 2.5-fold increase in the cytochrome c oxidase (COX) activity was observed in VHb-expressing cells relative to the control. In addition to this, exogenously added VHb in the assay mixture augmented COX activity.

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An Essential GTPase, Der, Containing Double GTP-binding Domains from Escherichia coli and Thermotoga maritima

Cited by 62 publications

References 20 publications

Structure-based design and screening of inhibitors for an essential bacterial GTPase, Der

Structure-based design and screening of inhibitors for an essential bacterial GTPase, Der

Analysis of GTPases carrying hydrophobic amino acid substitutions in lieu of the catalytic glutamine: Implications for GTP hydrolysis

Expression of Vitreoscilla hemoglobin enhances growth and levels of α-amylase in Schwanniomyces occidentalis

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