Lysyl-tRNA Synthetase of<i>Bacillus stearothermophilus</i>Molecular Cloning and Expression of the Gene

Takita, Teisuke; Shimizu, Naofumi; Sukata, Tokuo; Hashimoto, Shinichi; Akita, Eriko; Yokota, Toshihiko; Esaki, Nobuyoshi; Soda, Kenji; Inouye, Kuniyo; Tonomura, Ben’ichiro

doi:10.1271/bbb.64.432

Cited by 3 publications

(6 citation statements)

References 21 publications

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“…Proteins-The recombinant form of wild-type B.s LysRS was purified from E. coli BL21(DE3) cells by the method reported previously (25). The enzyme concentration was determined spectrophotometrically with ⑀ 280 of 70,600 M Ϫ1 cm Ϫ1 at pH 8.0, 25°C (25).…”

Section: Methodsmentioning

confidence: 99%

“…The enzyme concentration was determined spectrophotometrically with ⑀ 280 of 70,600 M Ϫ1 cm Ϫ1 at pH 8.0, 25°C (25). Streptomyces subtilisin inhibitor was a gift from Dr. B. Tonomura.…”

Section: Methodsmentioning

confidence: 99%

“…LysRS (25) ) of B.s. LysRS were designed to be expressed in the form with an Nterminal T7 Tag sequence as TAB-ND and CAT-CD, respectively (Table I).…”

Section: Methodsmentioning

confidence: 99%

“…By PCR reaction with pBLX45 containing the B.s. LysRS gene (25) and Pfu DNA polymerase, the genes corresponding to TAB-ND and CAT-CD were amplified with N2-and C2-primers and N1-and C1-primers, respectively. The amplified DNA fragments were digested with BamHI and inserted into the corresponding site of the pET11a expression vector.…”

Section: Methodsmentioning

confidence: 99%

“…TAB-ND and CAT-CD were purified in the same manner as the intact enzyme until the ammonium sulfate precipitation (60% saturation), except for omitting the heat treatment at 55°C (25). The solution was loaded to a DEAE-Toyopearl 650M column and eluted with a linear gradient of 0 -1 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

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Transition State Stabilization by the N-terminal Anticodon-binding Domain of Lysyl-tRNA Synthetase

Takita

Inouye

2002

Journal of Biological Chemistry

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Lysyl-tRNA synthetase from Bacillus stearothermophilus (B.s. LysRS) (EC 6.1.1.6) catalyzes aminoacylation of tRNA Lys with L-lysine, in which L-lysine was first activated with ATP to yield an enzyme (lysyladenylate complex), and then the lysine molecule was transferred from the complex to tRNA Lys . B.s. LysRS is a homodimeric enzyme with a subunit that consists of two domains, an N-terminal tRNA anticodon-binding domain (TAB-ND: Ser 1 -Pro 144 ) and a C-terminal Class IIspecific catalytic domain (CAT-CD: Lys 151 -Lys 493 ). CAT-CD alone retained catalytic activity, although at a low level; TAB-ND alone showed no activity. Size exclusion chromatography revealed that CAT-CD exists as a dimer, whereas TAB-ND was a monomer. The formation of a complex consisting of these domains was detected with the guidance of surface plasmon resonance. In accordance with this, the addition of TAB-ND to CAT-CD significantly enhanced both the L-lysine activation and the tRNA aminoacylation reactions. Kinetic analysis showed that deletion of TAB-ND resulted in a significant destabilization of the transition state of CAT-CD in the L-lysine activation reaction but had little effect on the ground state of substrate binding. A significant role of a cross-subunit interaction in the enzyme between TAB-ND and CAT-CD was proposed for the stabilization of the transition state in the L-lysine activation reaction. AaRS1 catalyzes the ligation of an amino acid to the cognate tRNA, generally according to the following (Reaction Scheme 1),where AA denotes the amino acid; E, aaRS; PPi, inorganic pyrophosphate; and E⅐AAϳAMP, an aaRS⅐aminoacyladenylate complex. Because the tRNA aminoacylation reaction is critical for the fidelity of translation of genetic information into the structure of a protein, aaRSs must have gained a high degree of substrate specificity for each heterogeneous substrate during evolution. Recent progress in x-ray crystallographic analysis has revealed that aaRSs can be classified into two groups according to their active site topology. Class I aaRSs possess a catalytic core composed of a Rossmann fold, whereas Class II aaRSs possess a catalytic core consisting of antiparallel ␤-sheets (2). AaRSs are considered to have emerged at an early stage in the development of the contemporary system of protein synthesis (3). In addition, it was reported that several isolated catalytic domains of aaRS retained full or part of the amino acid activation activity (4 -8). Together, these results have led to the proposal that the modern aaRS evolved from a primordial ancestor that had either of the two types of the classdefining domains, by getting the idiosyncratic region of each aaRS (3, 9, 10). In fact, the typical insertion in Class I aaRSs of the variable connective polypeptides (11) is responsible for the stabilization of the transition state of methionine activation as well as methionine transfer to the 3Ј end of tRNA in Escherichia coli MetRS (12), and also for the hydrolysis of misacylated Val-tRNA Ile in Thermus thermophilus IleRS (13...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…LysRS (25) ) of B.s. LysRS were designed to be expressed in the form with an Nterminal T7 Tag sequence as TAB-ND and CAT-CD, respectively (Table I).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Transition State Stabilization by the N-terminal Anticodon-binding Domain of Lysyl-tRNA Synthetase

Takita

Inouye

2002

Journal of Biological Chemistry

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Lysyl-tRNA Synthetase from Bacillus stearothermophilus: The Trp314 Residue is Shielded in a Non-polar Environment and is Responsible for the Fluorescence Changes Observed in the Amino Acid Activation Reaction

Takita

Nakagoshi

Inouye

et al. 2003

Journal of Molecular Biology

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Two Crystal Structures of Lysyl-tRNA Synthetase from Bacillus stearothermophilus in Complex with Lysyladenylate-Like Compounds: Insights into the Irreversible Formation of the Enzyme-Bound Adenylate of L-Lysine Hydroxamate

Sakurama¹,

Takita²,

Mikami³

et al. 2009

Journal of Biochemistry

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Aminoacyl-tRNA synthetase forms an enzyme-bound intermediate, aminoacyladenylate in the amino-acid activation reaction. This reaction is monitored by measuring the ATP-PPi exchange reason in which [(32)P]PPi is incorporated into ATP. We previously reported that L-lysine hydroxamate completely inhibited the L-lysine-dependent ATP-PPi exchange reaction catalysed by lysyl-tRNA synthetase from Bacillus stearothermophilus (BsLysRS). Several experiments suggested that BsLysRS can adenylate L-lysine hydroxamate, but the enzyme-bound lysyladenylate-like compound does not undergo the nucleophilic attack of PPi. This contrasts with the two reports for seryl-tRNA synthetase (SerRS): (i) L-serine hydroxamate was utilized by yeast SerRS as a substrate in the ATP-PPi exchange; and (ii) a seryladenylate-like compound was formed from L-serine hydroxamate in the crystal structure of Thermus thermophilus SerRS. To gain clues about the mechanistic difference, we have determined the crystal structures of two complexes of BsLysRS with the adenylate of L-lysine hydroxamate and with 5'-O-[N-(L-Lysyl)sulphamoyl] adenosine. The comparisons of the two BsLysRS structures and the above SerRS structure revealed the specific side-chain shift of Glu411 of BsLysRS in the complex with the adenylate of L-lysine hydroxamate. In support of other structural comparisons, the result suggested that Glu411 plays a key role in the arrangement of PPi for the nucleophilic attack.

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Lysyl-tRNA Synthetase ofBacillus stearothermophilusMolecular Cloning and Expression of the Gene

Cited by 3 publications

References 21 publications

Transition State Stabilization by the N-terminal Anticodon-binding Domain of Lysyl-tRNA Synthetase

Transition State Stabilization by the N-terminal Anticodon-binding Domain of Lysyl-tRNA Synthetase

Lysyl-tRNA Synthetase from Bacillus stearothermophilus: The Trp314 Residue is Shielded in a Non-polar Environment and is Responsible for the Fluorescence Changes Observed in the Amino Acid Activation Reaction

Two Crystal Structures of Lysyl-tRNA Synthetase from Bacillus stearothermophilus in Complex with Lysyladenylate-Like Compounds: Insights into the Irreversible Formation of the Enzyme-Bound Adenylate of L-Lysine Hydroxamate

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