Structures of Thermophilic and Mesophilic Adenylate Kinases from the Genus Methanococcus

Criswell, Angela; Bae, Euiyoung; Stec, Boguslaw; Konisky, J; Phillips, George N.

doi:10.1016/s0022-2836(03)00655-7

Cited by 59 publications

(77 citation statements)

References 58 publications

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“…Other structural features, such as high hydrophobic interactions, higher hydrogen bonding, amino acid substitutions, and high salt bridge content, among others (10,11,18,23,32,42,54), which were observed and studied in thermophilic proteins, could contribute to the thermostability of LipA and LipB, and have not been investigated here due to lack of the encoding gene sequence and crystal structure. However, the most profound effect on thermostability was noticed with the addition of 0.5 to 2 M ammonium sulfate to the enzyme solution prior to assaying activity, suggesting that hydrophobic interactions are important for the integrity of the LipA and LipB protein structures.…”

Section: Fig 4 Effect Of Phmentioning

confidence: 99%

Purification and Characterization of Two Highly Thermophilic Alkaline Lipases fromThermosyntropha lipolytica

Salameh

Wiegel

2007

Appl Environ Microbiol

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Two thermostable lipases were isolated and characterized from Thermosyntropha lipolytica DSM 11003, an anaerobic, thermophilic, alkali-tolerant bacterium which grows syntrophically with methanogens on lipids such as olive oil, utilizing only the liberated fatty acid moieties but not the glycerol. Lipases LipA and LipB were purified from culture supernatants to gel electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic interaction column chromatography. The apparent molecular masses of LipA and LipB determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 50 and 57 kDa, respectively. The temperature for maximal activity of LipA and LipB was around 96°C, which is, so far as is known, the highest temperature for maximal activity among lipases, and the pH optima for growth determined at 25°C (pH 25°C optima) were 9.4 and 9.6, respectively. LipA and LipB at 100°C and pH 25°C 8.0 retained 50% activity after 6 and 2 h of incubation, respectively. Both enzymes exhibited high activity with long-chain fatty acid glycerides, yielding maximum activity with trioleate (C 18:1 ) and, among the p-nitrophenyl esters, with p-nitrophenyl laurate. Hydrolysis of glycerol ester bonds occurred at positions 1 and 3. The activities of both lipases were totally inhibited by 10 mM phenylmethylsulfonyl fluoride and 10 mM EDTA. Metal analysis indicated that both LipA and LipB contain 1 Ca 2؉ and one Mn 2؉ ion per monomeric enzyme unit. The addition of 1 mM MnCl 2 to dialyzed enzyme preparations enhanced the activities at 96°C of both LipA and LipB by threefold and increased the durations of their thermal stability at 60°C and 75°C, respectively, by 4 h.

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Section: Fig 4 Effect Of Phmentioning

confidence: 99%

Purification and Characterization of Two Highly Thermophilic Alkaline Lipases fromThermosyntropha lipolytica

Salameh

Wiegel

2007

Appl Environ Microbiol

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“…Several unique structural features in thermophilic proteins have been observed (8). The modification of subtle intramolecular interactions such as hydrogen bonding (9), electrostatic interactions of ion pairs (10), and hydrophobic interactions (11) was proposed to be responsible for the increased thermal stability. On the other hand, the molecular mechanism of cold adaptation in psychrophilic proteins is still relatively unknown due to the limited number of available structures.…”

mentioning

confidence: 99%

Structures and Analysis of Highly Homologous Psychrophilic, Mesophilic, and Thermophilic Adenylate Kinases

Bae

Phillips

2004

Journal of Biological Chemistry

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188

221

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The crystal structures of adenylate kinases from the psychrophile Bacillus globisporus and the mesophile Bacillus subtilis have been solved and compared with that from the thermophile Bacillus stearothermophilus. This is the first example we know of where a trio of protein structures has been solved that have the same number of amino acids and a high level of identity (66 -74%) and yet come from organisms with different operating temperatures. The enzymes were characterized for their own thermal denaturation and inactivation, and they exhibited the same temperature preferences as their source organisms. The structures of the three highly homologous, dynamic proteins with different temperature-activity profiles provide an opportunity to explore a molecular mechanism of cold and heat adaptation. Their analysis suggests that the maintenance of the balance between stability and flexibility is crucial for proteins to function at their environmental temperatures, and it is achieved by the modification of intramolecular interactions in the process of temperature adaptation.There has recently been an increase in the discovery, isolation, and investigation of organisms inhabiting extreme environments (extremophiles) (1). Among these, organisms living at low and high temperatures (psychrophiles and thermophiles, respectively) have been of particular interest since proteins isolated from these organisms can function and remain stable at or near extreme temperatures (2, 3). These proteins have potential in industry directly or as models for engineering proteins from organisms living at moderate temperatures (mesophiles) because their unique properties such as activity at extreme temperatures and inactivity or instability at moderate temperatures are often desirable for industrial processes (4, 5). They have also drawn attention from academia because they provide a unique opportunity to study relationships between stability, dynamics, and function of proteins (6, 7). Therefore, research efforts have focused on comparative studies using proteins from temperature extremophiles and their mesophilic counterparts to illustrate a mechanism for temperature adaptation of proteins.Because of the relative ease of crystallizing thermophilic proteins, many structural studies have been performed to identify the molecular basis of heat adaptation by comparing thermophilic proteins with their mesophilic homologs. Several unique structural features in thermophilic proteins have been observed (8). The modification of subtle intramolecular interactions such as hydrogen bonding (9), electrostatic interactions of ion pairs (10), and hydrophobic interactions (11) was proposed to be responsible for the increased thermal stability. On the other hand, the molecular mechanism of cold adaptation in psychrophilic proteins is still relatively unknown due to the limited number of available structures. In fact, only a handful of crystal structures have been solved for psychrophilic proteins presumably because their thermolability and flexibility cause di...

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“…As expected, the LID domain of Zn-AK is in a more open conformation owing to the strong crystal packing interactions and has a different topology owing to the presence of the metal ion. One other structure of AK from Gram-negative bacteria-AK from A. aeolicus [38]-has been reported, as have several structures from Archaea-AK from Methanococcus voltae and Methanococcus thermolithotrophicus [43] (Gram negative) and AK from Methanococcus maripaludis [9,43] and Sulpholobus acidocaldarius (variable response to the Gram stain) [10]-but none of them contain a metal atom in their LID domain. The multiple sequence alignment of AK from D. gigas and these three reported AK is presented in Fig.…”

Section: Structural Comparison With Other Akmentioning

confidence: 99%

Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria

et al. 2010

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Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn 2? , Zn-AK; Co 2? , Co-AK; and Fe 2? , Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 Å , respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.

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Structures of Thermophilic and Mesophilic Adenylate Kinases from the Genus Methanococcus

Cited by 59 publications

References 58 publications

Purification and Characterization of Two Highly Thermophilic Alkaline Lipases fromThermosyntropha lipolytica

Purification and Characterization of Two Highly Thermophilic Alkaline Lipases fromThermosyntropha lipolytica

Structures and Analysis of Highly Homologous Psychrophilic, Mesophilic, and Thermophilic Adenylate Kinases

Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria

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