Takahisa Ohta

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.

Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing α-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47

Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVA I, an extracellular enzyme, and TVA II, an intracellular enzyme. Both enzymes hydrolyze pullulan to produce panose, and also hydrolyze cyclodextrins. We cloned and sequenced the TVA I gene. The TVA I gene consisted of 1833 base pairs, and the deduced primary structure was composed of 611 amino-acid residues, including an N-terminal signal sequence consisting of 29 amino-acid residues. The similarity between the amino-acid sequence of mature TVA I with those of other pullulan/cyclodextrin-hydrolyzing enzymes, such as TVA II and Bacillus stearothermophilus neopullulanase, was only 30%, although that of TVA II with neopullulanase was 48%. TVA II prefers specific small oligosaccharides and alpha- and beta-cyclodextrins. Whereas kcat/Km values of TVA I for pullulan were larger than that of TVA II, and TVA II could not hydrolyze starch completely. TVA II was inhibited by maltose, the hydrolysate of starch, which seems to be the reason for inefficient hydrolysis of starch. These kinetic properties indicate that TVA I and TVA II have differential physiological roles in sugar metabolism extracellularly and intracellularly, respectively.

Allosteric Activation of L-Lactate Dehydrogenase Analyzed by Hybrid Enzymes with Effector-sensitive and -insensitive Subunits

Subunit-hybrid enzymes of mutant tetrameric L-lactate dehydrogenases from Bifidobacterium longum were studied in an examination of the mechanism of allosteric activation by fructose 1,6-bisphosphate. We earlier developed an in vivo method for subunit hybridization in Escherichia coli and the hybrids formed were a mixture with different subunit compositions. The B. longum hybrids were separated by anion-exchange chromatography with a mutational tag. Hybrids formed between fructose 1,6-bisphosphate-desensitized subunits and wild-type subunits and also between fructose 1,6-bisphosphate-desensitized subunits and catalytically inactive subunits. Kinetic analyses of the hybrid enzymes showed that (i) those residues from two symmetrically related subunits that constituted the fructose 1,6-bisphosphate-binding site could bind fructose 1,6-bisphosphate and activate the enzyme only if intact, (ii) hybrids with only one functional fructose 1,6-bisphosphate-binding site were fully sensitive to fructose 1,6-bisphosphate, but the allosteric equilibrium had shifted partially, and (iii) activation by fructose 1,6-bisphosphate at the fructose 1,6-bisphosphate-binding site was transmitted to the active sites through a quaternary structural change, not through direct conformational change within a subunit. These results are evidence of the validity of the concerted allosteric model of this enzyme based on T- and R-state structures in the same crystal lattice proposed earlier.

Conformational change of tyrosyl-RNA synthetase induced by its specific transfer RNA☆

Abstract The tyrosyl-RNA synthetase which had been purified from bakers yeast was studied by circular dichroism. It was found that tyrosyl-RNA synthetase exhibited a marginal decrease in helical content only when the active enzyme was incubated with its specific transfer RNA. This conformational change is discussed in relation to enzyme function.

A water-soluble form of penicillin-binding protein 2 of Escherichia coli constructed by site-directed mutagenesis

Penicillin‐binding protein (PBP) 2 of Escherichia coli is located in the cytoplasmic membrane. The N‐terminal hydrophobic segment (31 amino acids, residues 15–45) of PBP2 was removed by a deletion in the PBP2 gene by site‐directed mutagenesis, resulting in the production of a water‐soluble form of PBP2 (called PBP2*). PBP2* retained the penicillin‐binding activity, was localized in the cytoplasm and was overproduced under the control of the lpp‐lac promoter. This indicates that the removed hydrophobic segment is an uncleaved signal sequence required for translocation of PBP2 across the cytoplasmic membrane, and also suggests that the segment anchors the protein to the membrane.

Identification of an allosteric site residue of a fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase of Thermus caldophilus GK24: production of a non-allosteric form by protein engineering

Fructose 1,6‐bisphosphate (Fru‐1,6‐P2)‐dependent L‐lactate dehydrogenase (LDH) of Thermus caldophilus GK24 can be converted to a Fru‐1,6‐P2‐independent form on modification with Arg‐specific reagents. After trypsin digestion of the modified LDH, a peptide containing a modified Arg residue was purified and sequenced, and the modified Arg residue was identified as Arg‐173. Subsequently, Arg‐173 was replaced with Gln to remove the positive charge by site‐directed mutagenesis. The mutant LDH was independent of Fru‐1,6‐P2, like non‐allosteric vertebrate LDHs. Thus, Arg‐173 was concluded to be located in the allosteric site and to be responsible for allosteric regulation of the LDH.

A 38 kDa precursor protein of aqualysin I (a thermophilic subtilisin‐type protease) with a C‐terminal extended sequence: its purification and in vitro processing

The precursor of aqualysin I, an extracellular subtilisin‐type protease produced by Thermus aquaticus, consists of four domains: an N‐terminal signal peptide, an N‐terminal pro‐sequence, a protease domain, and a C‐terminal extended sequence. In an Escherichia coli expression system for the aqualysin I gene, a 38 kDa precursor protein consisting of the protease domain and the C‐terminal extended sequence is accumulated in the membrane fraction and processed to a 28 kDa mature enzyme upon heat treatment at 65°C. The 38 kDa precursor protein is separated as a soluble form from denatured E. coli proteins after heat treatment. Accordingly, purification of the 38 kDa proaqualysin I was performed using chromatography. The purified precursor protein gave a single band on SDS‐polyacrylamide gels. The precursor protein exhibited proteolytic activity comparable to that of the mature enzyme. The purified precursor protein was processed to the mature enzyme upon heat treatment. The processing was inhibited by diisopropyl fluorophosphate. The processing rate increased upon either the addition of mature aqualysin I or upon an increase in the concentration of the precursor, suggesting that the cleavage of the C‐terminal extended sequence occurs through an intermolecular self‐processing mechanism.

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.

Involvement of the conserved histidine-188 residue in the L-lactate dehydrogenase from Thermus caldophilus GK24 in allosteric regulation by fructose 1,6-bisphosphate

The conserved histidine-188 residue of the L-lactate dehydrogenase of Thermus caldophilus GK 24, which is allosterically activated by fructose 1,6-bisphosphate, has been exchanged to phenylalanine by site-specific mutagenesis. In the mutant enzyme the strong stimulatory effect of fructose 1,6-bisphosphate is abolished. The analysis of the pH dependence of the activity indicates that the positive charge of the conserved His-188 residue is important for the interaction of the enzyme with the allosteric effector.

Production of thermophilic protease by glucose-controlled fed-batch culture of recombinant Escherichia coli

Abstract To achieve overproduction of the thermophilic protease aqualysin I (AQI), batch and fed-batch cultures of Escherichia coli TG1 transformed with an expression plasmid pAQN were investigated. The AQI gene, derived from Thermus aquaticus YT-1, was inserted under the control of the tac promoter in pAQN. During the fed-batch culture of recombinant E. coli TG1, acetic acid accumulated to a concentration of above 10 g/ l in the culture medium. This accumulation was closely related to the glucose concentration of the culture medium, and it affected not only cellular growth but also the rate of AQI production. When 12 g/ l of acetic acid was added to a batch culture at the time of IPTG induction, the rate dropped to 63% of the maximum value for batch culture without the addition of acetic acid. To suppress acetic acid accumulation, fed-batch cultures were carried out using an on-line glucose analyzer and HPLC unit to control the glucose feed rate, while the acetic acid concentration was monitored in real time. When the DO and glucose concentrations were maintained at 50% saturation and less than 0.3 g/ l , respectively, the acetic acid concentration remained at a low level. Under these culture conditions, 18 g/ l of cells and 33 kU/ml of enzymatically active AQI were obtained.