Haruhiko Masaki

Unusual genetic codes and a novel gene structure for tRNASeragy in starfish mitochondria! DNA

The nucleotide sequence of a 3849-bp fragment of starfish mitochondrial genome was determined. The genes for NADH dehydrogenase subunits 3, 4, 5, and COIII, and three kinds of (tRNA(UCNSer), tRNA(His), and tRNA(AGYSer) were identified by comparing with the genes of other animal mitochondria so far elucidated. The gene arrangement of starfish mitochondrial genome was different from those of vertebrate and insect mitochondrial genomes. Comparison of the protein-encoding nucleotide sequences of starfish mitochondria with those of other animal mitochondria suggested a unique genetic code in starfish mitochondrial genome; both AGA and AGG (arginine in the universal code) code for serine, AUA (isoleucine in the universal code but methionine in most mitochondrial systems) for isoleucine, and AAA (lysine) for asparagine. It was also inferred that these AGA and AGG codons are decoded by serine tRNA(AGYSer) originally corresponding to AGC and AGU codons. This situation is similar to the case of Drosophila mitochondrial genome. Variations in the use of AGA and AGG codons were discussed on the basis of the evolution of animals and decoding capacity of various tRNA(AGYSer) species possessing different sizes of the dihydrouridine (D) arm.

Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties

We have established an expression system of Trichoderma reesei cellulase genes using Aspergillus oryzae as a host. In this system, the expression of T. reesei cellulase genes were regulated under the control of A. oryzae Taka-amylase promoter and the cellulase genes were highly expressed when maltose was used as a main carbon source for inducer. The production of recombinant cellulases by A. oryzae transformants reached a maximum after 3-4 days of cultivation. In some cases, proteolysis of recombinant cellulases was observed in the late stage of cultivation. The recombinant cellulases were purified and characterized. The apparent molecular weights of recombinant cellulases were more or less larger than those of native enzymes. The optimal temperatures and pHs of recombinant cellulases were 50-70 degrees C and 4-5, respectively. Among the recombinant cellulases, endoglucanase I showed broad substrate specificities and high activity when compared with the other cellulases investigated here.

Replacement of an amino acid residue of cyclodextrin glucanotransferase of Bacillus ohbensis doubles the production of γ-cyclodextrin

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cyclodextrin (CD) from starch through an intramolecular transglucosylation reaction. To obtain a better understanding of the amylolytic and cyclization mechanisms of CGTase, and furthermore to improve the production of gamma-CD, mutant CGTases were constructed by site-directed mutagenesis of the CGTase gene of Bacillus ohbensis replacing Tyr at position 188 by 19 other amino acids. All mutant enzymes retained both starch-degrading and CD synthesizing activities to various extents. Among them, a mutant enzyme having Trp instead of Tyr-188 produced 15% of gamma-CD from soluble starch, which is about twice as much as the amount produced by the wild-type enzyme.

Cloning and sequencing of a cyclodextrin glucanotransferase gene from Bacillus ohbensis and its expression in Escherichia coli

SummaryA cyclcodextrin glucanotransferase (CGTase) gene of Bacillus ohbensis was cloned in Escherichia coli and the nucleotide sequence was determined. A single open reading frame (2112 bp) with a TTG codon as an initiator was identified that encodes a typical signal peptide of 29 amino acids followed by the mature enzyme (675 amino acids), of which the partial amino acid sequences of the N-terminal region and some lysyl-endopeptidase fragments were determined by Edman degradation. The CGTase gene was expressed in E. coli under control of the lac promoter only when the upstream region containing a long inverted repeat structure (located at −108 to −67 bp from the initiation codon) was deleted. Substitution of an ATG codon for the initiation TTG triplet doubled the expression of the CGTase gene in E. coli. Enzyme preparations purified from the culture supernatant of B. ohbensis and from the periplasmic fraction of the E. coli transformant exhibited the same molecular weight (Mr) and enzymatic properties as follows: Mr, 80 000; optimum pH for activity, 5.0 (and a suboptimum at 10.0); stability between pH 6.5 and 10.0; optimum temperature for activity, 55°C; and stability below 45°C. The yields of the products from starch as the substrate were 25% for β-and 5% for γ-cyclodextrin.

Cloning and sequencing of an α-glucosidase gene from Aspergillus niger and its expression in A. nidulans

We have cloned an extracellular alpha-glucosidase gene from Aspergillus niger with oligonucleotide probes synthesized on the basis of the determined peptide sequences. The nucleotide sequence revealed an open reading frame of 985 amino acids split with three introns, and the deduced amino acid sequence was nearly identical to that of the alpha-glucosidase previously determined. The cloned gene was introduced into Aspergillus nidulans, and its expression in the transformants was shown to be regulated by the carbon sources in the medium, suggesting that a common regulatory expression system is shared by these two species as is the case of other starch-degrading enzymes of Aspergillus species.

Correlation between cellulose binding and activity of cellulose-binding domain mutants of Humicola grisea cellobiohydrolase 1

The cellulose‐binding domains (CBDs) of fungal cellulases interact with crystalline cellulose through their hydrophobic flat surface formed by three conserved aromatic amino acid residues. To analyze the functional importance of these residues, we constructed CBD mutants of cellobiohydrolase 1 (CBH1) of the thermophilic fungus Humicola grisea, and examined their cellulose‐binding ability and enzymatic activities. High activity on crystalline cellulose correlated with high cellulose‐binding ability and was dependent on the combination and configuration of the three aromatic residues. Tyrosine works best in the middle of the flat surface, while tryptophan is the best residue in the two outer positions.

Over-expression of Tfam improves the mitochondrial disease phenotypes in a mouse model system

The phenotypes of mitochondrial diseases caused by mutations in mitochondrial DNA (mtDNA) have been proposed to be strictly regulated by the proportion of wild-type and pathogenically mutated mtDNAs. More specifically, it is thought that the onset of the disease phenotype occurs when cells cannot maintain the proper mitochondrial function because of an over-abundance of pathological mtDNA. Therapies that cause a decrease in the pathogenic mtDNA population have been proposed as a treatment for mitochondrial diseases, but these therapies are difficult to apply in practice. In this report, we present a novel concept: to improve mitochondrial disease phenotypes via an increase in the absolute copy number of the wild-type mtDNA population in pathogenic cells even when the relative proportion of mtDNA genotypes remains unchanged. We have succeeded in ameliorating the typical symptoms of mitochondrial disease in a model mouse line by the over-expression of the mitochondrial transcription factor A (Tfam) followed by an increase of the mtDNA copy number. This new concept should lead to the development of a novel therapeutic treatment for mitochondrial diseases.

Structural basis for sequence-dependent recognition of colicin E5 tRNase by mimicking the mRNA–tRNA interaction

Colicin E5—a tRNase toxin—specifically cleaves QUN (Q: queuosine) anticodons of the Escherichia coli tRNAs for Tyr, His, Asn and Asp. Here, we report the crystal structure of the C-terminal ribonuclease domain (CRD) of E5 complexed with a substrate analog, namely, dGpdUp, at a resolution of 1.9 Å. Thisstructure is the first to reveal the substrate recognition mechanism of sequence-specific ribonucleases. E5-CRD realized the strict recognition for both the guanine and uracil bases of dGpdUp forming Watson–Crick-type hydrogen bonds and ring stacking interactions, thus mimicking the codons of mRNAs to bind to tRNA anticodons. The docking model of E5-CRD with tRNA also suggests its substrate preference for tRNA over ssRNA. In addition, the structure of E5-CRD/dGpdUp along with the mutational analysis suggests that Arg33 may play an important role in the catalytic activity, and Lys25/Lys60 may also be involved without His in E5-CRD. Finally, the comparison of the structures of E5-CRD/dGpdUp and E5-CRD/ImmE5 (an inhibitor protein) complexes suggests that the binding mode of E5-CRD and ImmE5 mimics that of mRNA and tRNA; this may represent the evolutionary pathway of these proteins from the RNA–RNA interaction through the RNA–protein interaction of tRNA/E5-CRD.

Sequence-specific recognition of colicin E5, a tRNA-targeting ribonuclease

Colicin E5 is a novel Escherichia coli ribonuclease that specifically cleaves the anticodons of tRNATyr, tRNAHis, tRNAAsn and tRNAAsp. Since this activity is confined to its 115 amino acid long C-terminal domain (CRD), the recognition mechanism of E5-CRD is of great interest. The four tRNA substrates share the unique sequence UQU within their anticodon loops, and are cleaved between Q (modified base of G) and 3′ U. Synthetic minihelix RNAs corresponding to the substrate tRNAs were completely susceptible to E5-CRD and were cleaved in the same manner as the authentic tRNAs. The specificity determinant for E5-CRD was YGUN at −1 to +3 of the ‘anticodon’. The YGU is absolutely required and the extent of susceptibility of minihelices depends on N (third letter of the anticodon) in the order A > C > G > U accounting for the order of susceptibility tRNATyr > tRNAAsp > tRNAHis, tRNAAsn. Contrastingly, we showed that GpUp is the minimal substrate strictly retaining specificity to E5-CRD. The effect of contiguous nucleotides is inconsistent between the loop and linear RNAs, suggesting that nucleotide extension on each side of GpUp introduces a structural constraint, which is reduced by a specific loop structure formation that includes a 5′ pyrimidine and 3′ A.

A plasmid region encoding the active fragment and the inhibitor protein of colicin E3—CA38

Colicin E3 is a plasmid ColE3-CA38directed antibacterial protein composed of two polypeptides, protein A and protein B of M, 61000 and 10000, respectively [1,2]. After colicin E3 is adsorbed onto receptors in the outer membrane of sensitive cells, it finally inactivates ribosomes by cleaving the 16 S RNA in the 30s subunit at a specific site [3,4]. This ribonuclease activity is exclusively attributed to the T2A domain, the Cterminal part of protein A. Tryptic digestion of intact colicin E3 (i.e., the AB complex) gives the Tl fragment and the T2 complex. This complex consists of the active fragment T2A and the inhibitor protein B [5,6]. Models similar to the above have been proposed for colicin E2 and cloacin DF13 [7,8]. The inhibitor protein acts specifically on each bacteriocin, and is produced in a greater quantity than bacteriocin, and in this way may protect the host cell from lethality. Consequently, the inhibitor protein is usually referred to as ‘an immunity protein’ or ‘an immunity substance’ [1,9-121. Here, however, we simply refer to it as ‘protein B’. The amino acid sequences of E3-T2A fragment [13], E3-protein B [14] and DF13immunity protein [15] have been reported. or E3 seems to function adequately in both the induced and non-induced states (unpublished). Our attention has been directed toward the mechanisms of expression and regulation of colicinogenicity and immunity. Although recent studies have shown some of the details involved in molecular structures of colicin E2 and E3, little is known regarding the genetic constructions of ColE2-P9 and ColE3-CA38 plasmids. We now report the location and the nucleotide sequence of the DNA region encoding protein B and the T2A domain of protein A of colicin E3.