Seiki Kuramitsu

Intron in the gene for the ribosomal protein S16 of tobacco chloroplast and its conserved boundary sequences

SummaryThe nucleotide sequence of a tobacco chloroplast gene coding for a protein homologous to Escherichia coli ribosomal protein S16 (rps16) has been determined. It is located 690 bp upstream from the gene for tRNALys (UUU) on the same DNA strand. This gene has an 860 bp intron. The intron boundary sequences are similar to those of the tobacco chloroplast split genes for tRNAGly (UCC), tRNALys (UUU), tRNAVal (UAC) and ribosomal proteins L2 and S12. We classified these introns as a new group, designated group III, which is different from groups I and II proposed by Michel and Dujon (1983). The conserved boundary sequences of the group III introns are GTGCGNY at the 5′ ends and ATCNRY(N)YYAY at the 3′ ends. The rps16 coding region contains 255 bp (85 codons) and its deduced amino acid sequence shows 34% homology (46% conservative replacements) with that of E. coli ribosomal protein S16. The primary transcript of rps16 is 1.3 kb long.

[Arg292----Val] or [Arg292----Leu] mutation enhances the reactivity of Escherichia coli aspartate aminotransferase with aromatic amino acids.

Arg292 of E. coli aspartate aminotransferase was substituted with valine or leucine by site-directed mutagenesis. In comparison with the wild-type enzyme, either of the mutant enzymes showed a decrease by over 5 orders of magnitude of kcat/km values for aspartate and glutamate. This supports the contention that Arg292 is important for determining the specificity of this enzyme for dicarboxylic substrates. In contrast, mutant enzymes displayed a 5- to 10-fold increase in kcat/Km values for aromatic amino acids as substrates. Thus, introduction of an uncharged, hydrophobic side chain into position 292 leads to a striking alteration in substrate specificity of this enzyme, thereby improving catalytic efficiency toward aromatic amino acids.

Effects of replacement of tryptophan-140 by phenylalanine or glycine on the function of Escherichia coli aspartate aminotransferase.

Trp140 of E. coli aspartate aminotransferase has been converted to Phe or Gly by site-directed mutagenesis. As compared to the wild-type enzyme, either of the mutant enzymes showed 10- to 100-fold increase in Kms for natural dicarboxylic substrates, but did not show appreciable changes in Kms for aromatic substrates. Teh kcat values for dicarboxylic and aromatic substrates were greatly decreased by [Trp140----Gly] mutation, but were decreased to lesser extents by [Trp140----Phe] mutation. These findings suggested that N(1) of Trp140 may not be essential for catalysis, but may be partly involved in the binding of the distal carboxylate group of the dicarboxylic substrates.

Aromatic amino acid aminotransferase of Escherichiacoli: Nucleotide sequence of the tyrB gene

The tyrB gene of E. coli K-12, which encodes aromatic amino acid aminotransferase (EC 2.6.1.57) was cloned. The nucleotide sequence of about 2 kilobase pairs containing the gene was determined. The coding region of the tyrB gene and the deduced amino acid sequence revealed that the aromatic amino acid aminotransferase of E. coli is homologous with the aspartate aminotransferase.

Substitution of an arginyl residue for the active site lysyl residue (Lys258) of aspartate aminotransferase

The active site lysyl residue (Lys258) of E. coli aspartate amino transferase was substituted for an arginyl residue by oligonucleotide-directed, site-specific mutagenesis. The mutant enzyme was obviously unable to form an aldimine bond with pyridoxal 5-phosphate but firmly bound the coenzyme. The finding that the mutation did not lead to entire loss in the enzymic activity suggests that Lys258 may not be essential but auxiliary for enzymic catalysis. It is also conceived that the positive charge provided by Arg258 may contribute to the enzymic catalysis.

Molecular cloning and sequence analysis of full-length cDNA for mRNA of adrenodoxin oxidoreductase from bovine adrenal cortex☆

A full-length cDNA clone (pADR) for adrenodoxin reductase was isolated by means of immunological screening from a bovine adrenal poly(A)+ mRNA library. A cDNA insert of 1,973 base pairs in length encoded the entire amino acid sequence of the adrenodoxin reductase precursor protein, which consists of 492 amino acids including an extrapeptide of 32 amino acids at the NH2-terminus. The cloned cDNA contained the complete 3-noncoding region of 443 nucleotides including 59 nucleotides of poly(A) and 51 nucleotides in the 5-noncoding region. The amino acid sequences from the 33rd to 70th, the 117th to 123rd, the 207th to 225th, the 247th to 323rd, the 385th to 426th, the 444th to 461st, and the 487th to 492nd in the predicted structure were identical with those of the purified adrenodoxin reductase and its digested peptides, with only four exceptions.

Expression of bovine lung prostaglandin F synthase in Escherichia coli

The full-length bovine lung prostaglandin(PG) F synthase cDNA was constructed from partial cDNA clones and ligated into bacterial expression vector pUC8 to develop expression plasmid pUCPF1. This plasmid permitted the synthesis of bovine lung PGF synthase in Escherichia coli. The recombinant bacteria overproduced a 36-KDa protein that was recognized by anti-PGF synthase antibody, and the expressed protein was purified to apparent homogeneity. The expressed protein reduced not only carbonyl compounds including PGD2 and phenanthrenequinone but also PGH2; and the Km values for phenanthrenequinone, PGD2, and PGH2 of the expressed protein were 0.1, 100, and 8 microM, respectively, which are the same as those of the bovine lung PGF synthase. The protein produced PGF2 alpha from PGH2, and 9 alpha, 11 beta-PGF2 from PGD2 at different active sites. Moreover, the structure of the purified protein from Escherichia coli was essentially identical to that of the native enzyme in terms of C-terminal sequence, sulfhydryl groups, and CD spectra except that the nine amino acids provided by the lac Z gene of the vector were fused to the N-terminus. These results indicate that the expressed protein is essentially identical to bovine lung PGF synthase. We confirmed that PGF synthase is a dual function enzyme catalyzing the reduction of PGH2 and PGD2 on a single enzyme and that it has one binding site for NADPH.

Branched-Chain Amino Acid Aminotransferase from Escherichia Coli

The nucleotide sequence of the E.coli ilvE gene, which encodes branched-chain amino acid aminotranferase (IlvAT), was determined. The enzyme was overproduced by inserting the ilvE gene into a vector with a tac promotor.