A high-throughput and generic assay method for the determination of substrate specificities of thermophilic alpha-aminotransferases

For the determination of substrate specificities of thermophilic alpha-aminotransferases (AATs), a novel high-throughput assay method was developed. In this method, a thermophilic omega-aminotransferase (OAT) and a thermophilic aldehyde dehydrogenase (ALDH) are coupled to the AAT reaction. Glutamic acid is used as an amino group donor for the AAT reaction yielding 2-oxoglutalic acid. 2-Oxoglutalic acid produced by the AAT reaction is used as an amino group acceptor in the OAT reaction regenerating glutamic acid. The amino group donor of the OAT reaction is 5-aminopentanoic acid yielding pentanedioic acid semialdehyde which is oxidized by ALDH to pentanedioic acid with concomitant reduction of NADP(+) to NADPH. NADPH thus produced then reduces colorless tetrazolium salt into colored formazan. To construct such a reaction system, the genes for a thermophilic AAT, a thermophilic OAT and a thermophilic ALDH were cloned and expressed in Escherichia coli. These enzymes were subsequently purified and used to determine the activities of AAT for the synthesis of unnatural amino acids. This method allowed the clear detection of the AAT activities as it measures the increase in the absorbance on a low background absorbance reading.     

Synthesis of <Emphasis Type="SmallCaps">dl</Emphasis>-tryptophan by modified broad specificity amino acid racemase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IFO 12996

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive l-amino acids to dl-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of d-amino acids, especially d-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. l-Trp (20 mM) was successfully racemized, and the proportion of d-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial l-Trp.     

Interactions between homopolymeric amino acids (HPAAs)

Many human proteins contain consecutive amino acid repeats, known as homopolymeric amino acid (HPAA) tracts. Some inherited diseases are caused by proteins in which HPAAs are expanded to an excessive length. To this day, nine polyglutamine-related diseases and nine polyalanine-related diseases have been reported, including Huntington's disease and oculopharyngeal muscular dystrophy. In this study, potential HPAA-HPAA interactions were examined by yeast two-hybrid assays using HPAAs of approximately 30 residues in length. The results indicate that hydrophobic HPAAs interact with themselves and with other hydrophobic HPAAs. Previously, we reported that hydrophobic HPAAs formed large aggregates in COS-7 cells. Here, those HPAAs were shown to have significant interactions with each other, suggesting that hydrophobicity plays an important role in aggregation. Among the observed HPAA-HPAA interactions, the Ala28-Ala29 interaction was notable because polyalanine tracts of these lengths have been established to be pathogenic in several polyalanine-related diseases. By testing several constructs of different lengths, we clarified that polyalanine self-interacts at longer lengths (>23 residues) but not at shorter lengths (six to approximately 23 residues) in a yeast two-hybrid assay and a GST pulldown assay. This self-interaction was found to be SDS sensitive in SDS-PAGE and native-PAGE assays. Moreover, the intracellular localization of these long polyalanine tracts was also observed to be disturbed. Our results suggest that long tracts of polyalanine acquire SDS-sensitive self-association properties, which may be a prerequisite event for their abnormal folding. The misfolding of these tracts is thought to be a common molecular aspect underlying the pathogenesis of polyalanine-related diseases.     

Prostaglandin E2 production in ovarian cancer cell lines is regulated by cyclooxygenase-1, not cyclooxygenase-2

The significance of cyclooxygenase-2 (COX-2) expression in ovarian cancer has been discussed. In this study, we found increased expression of COX-1 mRNA and protein in three out of 10 ovarian cancer cell lines. Prostaglandin E 2 (PGE2) production was elevated in these three cell lines, but not in other seven cell lines. COX-2 protein was not detected in any of the cell lines. Cytosolic prostaglandin E synthase (cPGES) mRNA and protein were detected in all 10 cell lines. Membrane-associated PGES-1 (mPGES-1) was detected in some of the ovarian cell lines, but its presence did not correspond with PGE2 production. In contrast, mPGES-2 mRNA and protein were detected in all 10 cell lines. A nonselective COX inhibitor (indometacin) and a selective COX-1 inhibitor (SC-560) strongly inhibited PGE2 production by the three cell lines, while selective COX-2 inhibitors (NS-398 and rofecoxib) did not inhibit PGE2 production. In addition, increased expression of COX-1, not COX-2 protein was observed in the mass of ovarian cancer tissues from 22 patients when compared with that in normal tissue. These findings suggest that COX-1 might be a major enzyme regulating PGE2 production in ovarian cancer cells.     

Comparative analysis of the cytotoxicity of homopolymeric amino acids

Many human proteins have homopolymeric amino acid (HPAA) tracts, although the physiological significance or cellular effects of their presence is poorly understood. We previously reported that 20 kinds of HPAAs show characteristic intracellular localization and that among those, hydrophobic HPAAs aggregate strongly and form high molecular weight proteins when expressed in cultured cells. In this study, we investigated the cytotoxicity of 20 kinds of HPAAs. HPAA tracts of approximately 30 residues fused to the C-terminus of YFP were expressed in COS-7 cells. Cells expressing homopolymeric-Cys, -Ile, -Leu, and -Val showed low viability in Trypan Blue assay. Caspase-3 activity, which is usually upregulated in dying cells, was determined by measuring the cleavage of the peptide substrate Ac-DEVD-MCA and by detecting the cleaved active form of the caspase-3 by Western blotting. The activity of caspase-3 was drastically elevated in cells expressing those HPAAs which showed low viability in Trypan Blue assay. Interestingly, it was found that there is a correlation between the hydrophobicity of a single amino acid and the cytotoxicity of the corresponding HPAA as a homopolymer. These results indicate that the hydrophobicity of HPAAs may cause cytotoxicity.