Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Imbault, P.; Colas, Bernard; Sarantoglou, V.; Boulanger, Yves; Weil, Jacques-Henry

doi:10.1021/bi00523a031

Cited by 22 publications

(6 citation statements)

References 31 publications

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“…For this extracellular concentration, the intracellular concentration of leucine would be around 60 M in the cell, as deduced from accumulation studies carried out with L. lactis membrane vesicles (43). This concentration is higher than the K m for leucine from the cognate tRNA synthetase, which was determined to be 5 to 8 M in several bacteria (1,27,47). Thus the maintenance of an intracellular pool of 60 M leucine should ensure normal growth.…”

Section: Discussionmentioning

confidence: 98%

Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis

et al. 1997

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The ␣-acetolactate decarboxylase gene aldB is clustered with the genes for the branched-chain amino acids (BCAA) in Lactococcus lactis subsp. lactis. It can be transcribed with BCAA genes under isoleucine regulation or independently of BCAA synthesis under the control of its own promoter. The product of aldB is responsible for leucine sensibility under valine starvation. In the presence of more than 10 M leucine, the ␣-acetolactate produced by the biosynthetic acetohydroxy acid synthase IlvBN is transformed to acetoin by AldB and, consequently, is not available for valine synthesis. AldB is also involved in acetoin formation in the 2,3-butanediol pathway, initiated by the catabolic acetolactate synthase, AlsS. The differences in the genetic organization, the expression, and the kinetics parameters of these enzymes between L. lactis and Klebsiella terrigena, Bacillus subtilis, or Leuconostoc oenos suggest that this pathway plays a different role in the metabolism in these bacteria. Thus, the ␣-acetolactate decarboxylase from L. lactis plays a dual role in the cell: (i) as key regulator of valine and leucine biosynthesis, by controlling the acetolactate flux by a shift to catabolism; and (ii) as an enzyme catalyzing the second step of the 2,3-butanediol pathway.Synthesis of the three branched-chain amino acids (BCAA), leucine, isoleucine, and valine, has been studied in detail in organisms as diverse as bacteria, fungi, and plants (for reviews, see references 8, 32, 57, and 58). A particular feature of this synthesis is that it is carried out, in part, by the same enzymes for the three amino acids (Fig. 1). However, the relative amounts of the three amino acids in cell proteins are not the same, since leucine, valine, and isoleucine represent 39, 36, and 25%, respectively, of the total BCAA in Escherichia coli (40). Regulation of the BCAA synthesis is therefore necessarily complex. A global regulation model has been established only for E. coli (58). Two levels of regulation, concerning gene transcription and enzyme inhibition, have previously been described.E. coli genes which encode BCAA synthesis enzymes form three clusters and are organized in five transcription units (2). Initiation of transcription of all the genes except ilvC is regulated by nonspecific, pleiotropic regulators responding to amino acid starvation and different changes in the medium (58). In addition to this control, a mechanism of transcriptional attenuation dependent on the synthesis of a leader peptide as described first for the tryptophan operon (33) negatively regulates ilvBN, ilvGMEDA, and leuABCD. These mechanisms allow the expression of the enzymes necessary for BCAA synthesis when needed.Activity of several BCAA-synthesizing enzymes is regulated by retroinhibition (Fig. 1). This control is simple in the case of leucine and isoleucine, since each amino acid inhibits an enzyme specific for an early step of its synthesis (LeuA and IlvA, respectively [53,57]). In contrast, the first enzyme involved in valine synthesis is also required fo...

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Section: Discussionmentioning

confidence: 98%

Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis

et al. 1997

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“…In the leucylation reaction, for the three substrates of hmLeuRS‐B ( E. coli tRNA 1 Leu used in the assay), the k cat value of hmLeuRS‐B is 4.8±0.5×10 −2 s −1 , about 3‐fold lower than that in the ATP‐PPi exchange reaction. This value is rather low as compared with that of E. coli LeuRS (3.3 s −1 ) [24], A. aeolicus LeuRS (0.39 s −1 ) [11], P. vulgaris cytoplasmic LeuRS (2.05 s −1 ) [25], Saccharomyces cerevisiae cytoplasmic LeuRS (3.3 s −1 ) [26], plant P. vulgaris chloroplastic LeuRS (3.15 s −1 ) [27], and Euglena gracilis chloroplastic LeuRS (2 s −1 ) [28]. The K m values for leucine and ATP were 40 and 1192 μM, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…C represents the consensus sequence of these seven LeuRSs. [28]. The K m values for leucine and ATP were 40 and 1192 WM, respectively.…”

Section: Activity Assay Of Hmleurs-bmentioning

confidence: 94%

The processing of human mitochondrial leucyl‐tRNA synthetase in the insect cells

Yao

Wang

et al. 2003

FEBS Letters

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A His-tagged full-length cDNA of human mitochondrial leucyl-tRNA synthetase was expressed in a baculovirus system. The N-terminal sequence of the enzyme isolated from the mitochondria of insect cells was found to be IYSATGKWT-KEYTL, indicating that the mitochondrial targeting signal peptide was cleaved between Ser39 and Ile40 after the enzyme precursor was translocated into mitochondria. The enzyme puri¢ed from mitochondria catalyzed the leucylation of Escherichia coli tRNA Leu 1 (CAG) and Aquifex aeolicus tRNA Leu (GAG) with higher catalytic activity in the leucylation of E. coli tRNA Leu than that previously expressed in E. coli without the N-terminal 21 residues. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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“…exception from the closer resemblance of cytoplasmic and mitochondrial than cytoplasmic or mitochondrial and prokaryotic enzymes is the association of carbohydrates with mitochondrial phenylalanyl-tRNA synthetases (Gabius et al, 1983c). The functional role of carbohydrates that are also found with Euglena gracilis chloroplastic leucyl-tRNA synthetase (Imbault et al, 1981) and cytoplasmic rat liver arginyl-and lysyl-tRNA synthetases (Dang et al, 1982) is unknown.…”

Section: Discussionmentioning

confidence: 99%

Evolutionary aspects of accuracy of phenylalanyl-tRNA synthetase. Accuracy of fungal and animal mitochondrial enzymes and their relationship to their cytoplasmic counterparts and a prokaryotic enzyme

Gabius¹,

Engelhardt²,

Fr³

et al. 1983

Biochemistry

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Phenylalanyl-tRNA synthetases from mitochondria of yeast and hen liver resemble their corresponding cytoplasmic counterparts. Whereas slight intraspecies differences at the amino acid binding site, reflecting variations in the structures of these distinct enzymes, are exploitable by phenylalanine analogues, no intraspecies difference can be noted for the strategies to achieve the high fidelity of protein synthesis. While the yeast mitochondrial enzyme follows the pathway of posttransfer proofreading, the hen liver mitochondrial enzyme uses a tRNA-dependent pretransfer proofreading in the case of the natural amino acids. The accuracy of mitochondrial phenylalanyl-tRNA synthetases appears to be even better than the accuracy of the corresponding cytoplasmic enzymes. Interspecies rather than intraspecies differences for the functional role of certain amino acid residues of the enzymes further indicate the close relationship of the intracellular heterotopic isoenzymes. By use of a highly sensitive immunospotting procedure, common antigenic determinants are detected only within the enzymes from the two intracellular compartments of the same organism. The results suggest the origin of the cytoplasm-mitochondrion isoenzyme pair by independent gene duplication of the ancestral nuclear gene. A similarity of mitochondrial enzymes to the phenylalanyl-tRNA synthetase from Escherichia coli is not observed.

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Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Cited by 22 publications

References 31 publications

Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis

Dual role of alpha-acetolactate decarboxylase in Lactococcus lactis subsp. lactis

The processing of human mitochondrial leucyl‐tRNA synthetase in the insect cells

Evolutionary aspects of accuracy of phenylalanyl-tRNA synthetase. Accuracy of fungal and animal mitochondrial enzymes and their relationship to their cytoplasmic counterparts and a prokaryotic enzyme

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