Proton magnetic resonance spectra of tRNA-Met-f from Thermus thermophilus.

220 MHz proton magnetic resonance spectra of tRNAs in bulk and tRNA-Met-f from Thermus thermophilus have been measured and compared with those of tRNAs from E. coli. Temperature dependences and chemical shift positions of the bulk tRNAs are well explained by the difference in their GC contents. It is known that the base sequence of the double helical regions in the cloverleaf structure of T. thermophilus tRNA-Met-f is different from that of E. coli tRNA-Met-f only at two positions in TpsiCarm; one more C:G pair is contained instead of a U:G pair of E. coli tRNA-Met-f and a C:G pair of E. coli is replaced by a G:C pair. In spite of the resembrance in the base sequences, nmr patterns around 13 ppm are fairly different from each other. The difference is discussed in relation with their tertiary structures and with the origin of chemical shift displacements.     

C-type cytochromes isloated from an extreme thermophile, Thermus thermophilus HB8.

Two cytochromes of the C-type, c-554 and c-549, were isolated from the soluble fraction of an extreme thermophile, Thermus thermophilus HB8. Highly purified cytochrome c-554 had absorption maxima at 554, 522, and 417 nm in the reduced state, and at 410 nm in the oxidized state. The alpha-band of the reduced state resembled that of "split-alpha" cytochromes. The isoelectric point was at pH 4.9, and the molecular weight was about 29,000. Cytochrome c-549, partially purified, had absorption maxima a6 549,520, and 416 nm in the reduced form, and at 408 nm in the oxidized form. The molecular weight was about 25,000. Both were slowly auto-oxidizable, and did not combine with CO.     

Acidic glycosphingolipids of human amnion

Six major acidic glycosphingolipids were isolated from human amnion using DEAE Sephadex A-25 and silica beads column chromatography. The structures of these glycosphingolipids were determined by methylation analysis, TLC immunostaining and/or negative ion FAB-MS, and were concluded to be II3 alpha NeuAcLacCer(GM3), IV3 alpha NeuAcnLc4-Cer (sialyl[alpha 2-3]paragloboside), IV6 alpha NeuAcnLc4Cer (sialyl[alpha 2-6]paragloboside), IV3 alpha NeuAcIII4 alpha FucLc4Cer (sialyl Lea), VI3 alpha NeuAcnLc6Cer (i-ganglioside) and II3 alpha (NeuAc alpha 2----8NeuAc)LacCer (GD3). In addition, several minor glycosphingolipids were detected with specific monoclonal antibodies, including glycolipids with NeuAc alpha 2-3Gal beta 1-4GlcNAc-beta 1- or NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1- determinant. Our results show that the glycosphingolipids of human amnion are characterized by having mainly type II chain analogues and onco-fetal antigens.     

Molecular cloning and nucleotide sequence of 3-isopropylmalate dehydrogenase gene (leuB) from an extreme thermophile, Thermus aquaticus YT-1

A gene (leuB) coding for 3-isopropylmalate dehydrogenase [EC 1.1.1.85] from an extreme thermophile, Thermus aquaticus YT-1 was cloned in Escherichia coli and the nucleotide sequence was determined. It contains an open reading frame of 1,035 bp encoding 344 amino acid residues. The homology with that from T. thermophilus HB8 is 87.0% in nucleotide and 91.3% in amino acid sequences. No overlapped gene was found in the present leuB gene, in contrast to the previous prediction that Thermus leuD gene is overlapped with leuB [Croft et al. (1987) Mol. Gen. Genet. 210, 490-497]. Substitutions in the primary structure which are unique for the thermophile sequences are discussed in relation to the unusual stability of the thermophile dehydrogenase based on amino acid sequence comparison of 9 microorganisms including thermophiles and mesophiles.     

Three-dimensional structure of a highly thermostable enzyme, 3-isopropylmalate dehydrogenase of Thermus thermophilus at 2.2 A resolution.

The three-dimensional structure of the highly thermostable 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus has been determined by the multiple isomorphous replacement method and refined to 2.2 A resolution. The final R-factor is 0.185 for 20,307 reflections. The crystal asymmetric unit has one subunit consisting of 345 amino acid residues. The polypeptide chain of this subunit is folded into two domains (first and second domains) with parallel alpha/beta motifs. The domains are similar in their conformations and folding topologies, but differ from those of the NAD-binding domains of such well-known enzymes as the alcohol and lactate dehydrogenases. A beta-strand that is a part of the long arm-like polypeptide protruding from the second domain comes into contact with another subunit and contributes to the formation of an isologous dimer with a crystallographic 2-fold symmetry. Close subunit contacts are also present at two alpha-helices in the second domain. These helices strongly interact hydrophobically with the corresponding helices of the other subunit to form a hydrophobic core at the center of the dimer. Two large pockets that exist between the first domain of one subunit and the second domain of the other include the amino acid residues responsible for substrate binding. These results indicate that the dimeric form is essential for the IPMDH to express enzymatic activity and that the close subunit contact at the hydrophobic core is important for the thermal stability of the enzyme.     

Cloning and characterization of the CYS3 (CYI1) gene of Saccharomyces cerevisiae.

A DNA fragment containing the Saccharomyces cerevisiae CYS3 (CYI1) gene was cloned. The clone had a single open reading frame of 1,182 bp (394 amino acid residues). By comparison of the deduced amino acid sequence with the N-terminal amino acid sequence of cystathionine gamma-lyase, CYS3 (CYI1) was concluded to be the structural gene for this enzyme. In addition, the deduced sequence showed homology with the following enzymes: rat cystathionine gamma-lyase (41%), Escherichia coli cystathionine gamma-synthase (36%), and cystathionine beta-lyase (25%). The N-terminal half of it was homologous (39%) with the N-terminal half of S. cerevisiae O-acetylserine and O-acetylhomoserine sulfhydrylase. The cloned CYS3 (CYI1) gene marginally complemented the E. coli metB mutation (cystathionine gamma-synthase deficiency) and conferred cystathionine gamma-synthase activity as well as cystathionine gamma-lyase activity to E. coli; cystathionine gamma-synthase activity was detected when O-succinylhomoserine but not O-acetylhomoserine was used as substrate. We therefore conclude that S. cerevisiae cystathionine gamma-lyase and E. coli cystathionine gamma-synthase are homologous in both structure and in vitro function and propose that their different in vivo functions are due to the unavailability of O-succinylhomoserine in S. cerevisiae and the scarceness of cystathionine in E. coli.     

Half-site recombinations mediated by yeast site-specific recombinases Flp and R.

The Flp recombinase of Saccharomyces cerevisae and the related R recombinase of Zygosaccharomyces rouxii can efficiently catalyze strand cleavage and strand exchange reactions in half recombination sites. A half-site consists of one recombinase binding element, a recombinase cleavage site on one strand and a 5' spacer hydroxyl group on the other that can initiate the strand exchange reaction. We have studied the various types of strand exchanges that half-sites can participate in. Reaction between a left half-site and a right half-site generates a full recombination site. Strand transfer between two left half-sites or between two right half-sites produces pseudo-full-sites. Strand transfer within a half-site results in a stem-loop or hairpin product. The half-site strand transfer reaction is fairly indifferent to the spacer sequence of the substrate per se and is less sensitive to variations in spacer lengths than a full-site recombination reaction. The optimal spacer length of eight to ten nucleotides observed for the Flp half-site reaction likely permits the most productive catalytic interactions between two Flp monomers bound to each of two partner half-sites. When reacted with a full-site, the half-site can give rise to a normal or reverse recombinant, corresponding to homologous or non-homologous alignments of the spacer sequences during substrate synapsis. The contrary recombination (resulting from non-homologous spacer alignment), whose level is low relative to normal recombination, is partly suppressed when the half-site spacer ends in a 5'-phosphate rather than a 5'-hydroxyl group. Thus, the early steps of recombination, namely synapsis and initial stand transfer, are not dependent on complete spacer homology between the two recombining substrates. The selection of properly aligned substrate partners must occur at the homology dependent branch migration step. In reactions containing a mixture of Flp and R half-sites, Flp and R catalyze strand transfer, almost exclusively, within or between their respective cognate substrates. However, under conditions where self-crosses are inhibited, strand exchange between a Flp half-site and an R half-site appears to be stimulated by a combination of R and Flp.     

Production and characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1.

N-Acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1 was inducibly produced by D isomers of N-acetylglutamate, glutamate, aspartate, and asparagine. The enzyme has been purified to homogeneity by DEAE-cellulose, (NH4)2SO4 fractionation, and chromatofocusing followed by gel filtration on a Sephadex G-100 column. The enzyme was a monomer with molecular weight of 55,000. The enzyme activity was optimal at pH 6.5 to 7.5 and 45 degrees C. The isoelectric point and the pH stability were 8.8 and 9.0, respectively. N-Formyl, N-acetyl, N-butyryl, N-propionyl, N-chloroacetyl derivatives of D-glutamate and glycyl-D-glutamate were substrates for the enzyme. At pH 6.5 in 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at 30 degrees C, a Km of 6.67 mM and a Vmax of 662 mumol/min/mg of protein for N-acetyl-D-glutamate were obtained. None of the metal ions stimulated the enzyme activity. Na+, K+, Mg2+, and Ba2+ acted as stabilizers. Hg2+, Cu2+, Zn2+, Fe3+, and EDTA were strongly inhibitory.