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Structure of the phenylalanyl-tRNA synthetase genes from Thermus thermophilus HB8 and their expression in Escherichia coli.

ABSTRACT: A 4459 bp long BamHI restriction fragment containing the two genes for the Thermus thermophilus HB8 phenylalanyl-tRNA synthetase was cloned in Escherichia coli and its nucleotide sequence was determined. The genes pheS and pheT encode the alpha- and beta-subunits with a molecular weight of 39 and 87 kD, respectively. Three conserved sequence motifs typical for class II tRNA synthetases occur in the alpha-subunit. Secondary structure predictions indicate that an arm composed of two anti-parallel alpha-helices similar to that reported for the E.coli seryl-tRNA synthetase may be present in its N-terminal portion. In the beta-subunit clusters of hydrophilic amino acids and a leucine zipper motif were identified, and several pronounced alpha-helical regions were predicted. The particular arginine and lysine residues in the N-terminal portion of the beta-subunit, which were found to participate in tRNA binding in the yeast and E.coli PheRSs, have their counterparts in the T.thermophilus protein. The 5'-portion of an open reading frame downstream of pheT was found and codes for a yet unidentified, extremely hydrophobic peptide. The pheST genes are presumably cotranscribed and translationally coupled. A novel type of a putative transcriptional terminator in Thermus species was identified immediately downstream of pheT and other Thermus genes. The genes pheS and pheST were expressed in E.coli.

SUBMITTER: Kreutzer R

PROVIDER: S-EPMC334122 | biostudies-other | 1992 Aug

REPOSITORIES: biostudies-other

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Structure of the phenylalanyl-tRNA synthetase genes from Thermus thermophilus HB8 and their expression in Escherichia coli.

Kreutzer R R Kruft V V Bobkova E V EV Lavrik O I OI Sprinzl M M

Nucleic acids research 19920801 16

A 4459 bp long BamHI restriction fragment containing the two genes for the Thermus thermophilus HB8 phenylalanyl-tRNA synthetase was cloned in Escherichia coli and its nucleotide sequence was determined. The genes pheS and pheT encode the alpha- and beta-subunits with a molecular weight of 39 and 87 kD, respectively. Three conserved sequence motifs typical for class II tRNA synthetases occur in the alpha-subunit. Secondary structure predictions indicate that an arm composed of two anti-parallel ...[more]

PMID: 1508711

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Project description:BackgroundMethylenetetrahydrofolate reductase (MTHFR) is one of the enzymes involved in homocysteine metabolism. Despite considerable genetic and clinical attention, the reaction mechanism and regulation of this enzyme are not fully understood because of difficult production and poor stability. While recombinant enzymes from thermophilic organisms are often stable and easy to prepare, properties of thermostable MTHFRs have not yet been reported.Methodology/principal findingsMTHFR from Thermus thermophilus HB8, a homologue of Escherichia coli MetF, has been expressed in E. coli and purified. The purified MTHFR was chiefly obtained as a heterodimer of apo- and holo-subunits, that is, one flavin adenine dinucleotide (FAD) prosthetic group bound per dimer. The crystal structure of the holo-subunit was quite similar to the β(8)α(8) barrel of E. coli MTHFR, while that of the apo-subunit was a previously unobserved closed form. In addition, the intersubunit interface of the dimer in the crystals was different from any of the subunit interfaces of the tetramer of E. coli MTHFR. Free FAD could be incorporated into the apo-subunit of the purified Thermus enzyme after purification, forming a homodimer of holo-subunits. Comparison of the crystal structures of the heterodimer and the homodimer revealed different intersubunit interfaces, indicating a large conformational change upon FAD binding. Most of the biochemical properties of the heterodimer and the homodimer were the same, except that the homodimer showed ≈50% activity per FAD-bound subunit in folate-dependent reactions.Conclusions/significanceThe different intersubunit interfaces and rearrangement of subunits of Thermus MTHFR may be related to human enzyme properties, such as the allosteric regulation by S-adenosylmethionine and the enhanced instability of the Ala222Val mutant upon loss of FAD. Whereas E. coli MTHFR was the only structural model for human MTHFR to date, our findings suggest that Thermus MTHFR will be another useful model for this important enzyme.

Dataset Information

Structure of the phenylalanyl-tRNA synthetase genes from Thermus thermophilus HB8 and their expression in Escherichia coli.

Publications

Structure of the phenylalanyl-tRNA synthetase genes from Thermus thermophilus HB8 and their expression in Escherichia coli.

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