Transaminase mutant

This colorimetric assay was used in a study by Matcham et al. for detechon of mutants with higher tolerance toward (S)-methoxyisopropylamine ((S)-MOIPA) and higher thermal and chemical stability [60]. Due to a slightly increased product con-centrahon after adding higher amounts of enzyme, it was necessary to improve the product tolerance of the enzyme. Error-prone PCR and several rounds of enzyme modification created a mutant with the required properhes. At the end of the reachon the soluhon contained not only the desired product, (S)-MOIPA, but also acetone. To shift the equilibrium toward the product side by evaporahng acetone by-product, a transaminase mutant thermally stable up to 50 °C was found after five rounds of mutation and used in this process. The hnal yield of (S)-MOIPA about 2mM was obtained after 7h reaction hme with 5g/l of the transaminase at 50°C under vacuum. 

A possible explanation for the superiority of the amino donor, L-aspartic add, has come from studies carried out on mutants of E. coli, in which only one of the three transaminases that are found in E. coli are present. It is believed that a branched chain transaminase, an aromatic amino add transaminase and an aspartate phenylalanine aspartase can be present in E. coli. The reaction of each of these mutants with different amino donors gave results which indicated that branched chain transminase and aromatic amino add transminase containing mutants were not able to proceed to high levels of conversion of phenylpyruvic add to L-phenylalanine. However, aspartate phenylalanine transaminase containing mutants were able to yield 98% conversion on 100 mmol l 1 phenylpyruvic acid. The explanation for this is probably that both branched chain transaminase and aromatic amino acid transminase are feedback inhibited by L-phenylalanine, whereas aspartate phenylalanine transaminase is not inhibited by L-phenylalanine. In addition, since oxaloacetate, which is produced when aspartic add is used as the amino donor, is readily converted to pyruvic add, no feedback inhibition involving oxaloacetate occurs. The reason for low conversion yield of some E. coli strains might be that these E. cdi strains are defident in the aspartate phenylalanine transaminase. 

Although K258A mutant is considered to be a dead enzyme, it has shown a small residual transaminase activity. The stereochemical fidelity of the K258A mutant enzyme was examined by measuring the residual transamination reaction in which the labilization of the pro-S C-4 hydrogen of PMP was monitored.42 During the normal transamination reaction, it was observed that the a-proton of the substrate was trasferred to the pro-S C-4 positionof PMP and the same proton was removed from the pro-S C-4 position then transferred to the second substrate, a part of the 1,3-prototropic shift in the transamination... 

Try-70 is conserved in all transaminases1 3 these transaminases utilize Glu a-KG pair as substrates. X-ray studies have shown that Tyr-70 interacts with the phosphate part of the coenzyme. Y70 F mutant was prepared to examine whether or not Tyr-70 could be a catalytic base. The Y70F mutant showed 15% of the activity of the wild-type enzyme thus, it was quite unlikely that Tyr-70 was the catalytic base. However, Tyr-70 was involved in the interaction with the coenzyme, since the affinity towards the coenzyme was significantly reduced in Y70 F mutant.49-513 The equilibrium dissociation constants for the PMP holoenzyme complex were 1.3 nM and 30 nM for the wild-type and mutant... 

HBe-minus-HBV mutant With fluctuating increases in transaminases, the reaction to HBV DNA can nevertheless be positive in the presence of anti-HBe. HBeAg formation is not induced. These patients are particularly endangered by the rapid develoj> ment of CAH and cirrhosis, (s. pp 114, 424) (s. tab. 22.3)... 

HBe-minus phase Predominance of minus mutants which can no longer synthesize HBeAg (so-called precore mutants). HBeAg is therefore negative, transaminases are stiU more than twice the normal value, HBV-DNA levels are elevated (> 10 copies/ml). ... 

HBeAg-negative patients possess precore (minus) mutants. They show an initial response rate of up to 90% there is no sign of flare-up . The aim of therapy is normalization of the transaminases and a loss of HBV DNA after > 12 months. After IFNa therapy has been stopped, reactivation usually occurs, even after several years. Only 15-25% of cases achieve sustained response. For this reason, an IFN therapy lasting 12-24... 

Relapse Reactivation is often associated with the presence of wildtype HBV (80-85%) or the occurrence of precore mutants (15-20%). This event (increase of transaminases, evidence of replication markers such as HBeAg and HBV DNA, positive IgM anti-HBC, pronounced interface hepatitis) develops in 15-20% of cases 1-3 years after IFNa therapy has been stopped. In such cases the question then arises of whether to apply lamivudine. This antiviral agent is administered in cases with an unfavourable constellation or contraindication regarding IFNa, or if the patient refuses IFN. 

Viral mutants As the duration of lamivudine therapy increases, the number of mutants rises, especially those resulting from YMDD-polymerase mutation. Viral mutants are found in about 20% of cases after 1 year, in 35-40% after 2 years and in 60-70% after 4 years. (159) Clinically, lamivudine resistance is recognizable due to an increase of transaminases and a recurrence of vir-aemia. Risk factors include high GPT (ALT) values, an elevated HBV-DNA level and a pathological body mass index. In the case of resistance, the use of adefovir is recommended, since this nucleoside is also efficacious against HBV mutants. 

Dosage is 10 mg/day orally duration of treatment is 48 weeks. The effect of ADV is not impaired by a high viral load. In HBeAg-positive patients, it results in elimination of HBV DNA in 20-25%, seroconversion in 15-20% and normalization of transaminases in 45-50% of cases. In HBeAg-negative patients, ADV shows a loss of HBV DNA in approx. 50% and normalization of GPT (ALT) in 70-75% of cases. In addition, a considerable improvement in histology was achieved. Viral resistance (due to mutants) was observed in < 3% of cases after 3 years. ADV can also be applied in decompensated cirrhosis. Tolerance is good. As with lamivudine, abrupt discontinuation may lead to acute exacerbation (in about 25%). (167, I8l, 193, 205, 206)... 

Entecavir is a guanosine nucleoside with strong antiviral activity. It is also effective in lamivudine-resistant patients. After 48 weeks of treatment comprising a daily dose of 0.5 or 1.0 mg, there was elimination of HBV DNA in 25 — 30% and normalization of the transaminases in 60—70% of lamivudine-resistant patients. Tolerance is good. Entecavir-induced mutants were not detected. (173)... 

The X-ray induced mutant C-2A was cultured and harvested as described earlier (10). DOVA-transaminase was purified as demonstrated in (11). The preparation of tRNA-fractions and the corresponding ligase preparation as well as the run-off fraction, containing the transaminases, ALA-dehydratase and porphobilinogenase were performed following (12). G-1-SA-pyrrole was formed by the condensation of the compound with ethylacetoacetate as described in (13). Protochlorophyllide (Pchlide) was isolated following (14). The separation into divinyl- and monovinyl-Pchlide was performed as described in (15). 

Similar experiments were carried out with tetracycline instead of streptomycin, and very similar results were obtained [103]. In addition to the argR strain, an argR strain was examined, as well as another mutant strain (grgR ) with diminished repressibility and diminished derepressibility [103]. For these strains, too, streptomycin and tetracycline behaved alike. Data obtained with tetracycline are listed in Table II. With respect to acetylornithine 5-transaminase and arginino-succinase, for the argR strain, the differential rates are seen to be... 

Three relevant enz3une activities have been demonstrated in E. coli extracts. These are an acetylase which forms JV-acetylglutamate from glutamic acid and acetyl CoA 129), a transaminase which forms N -acetylornithine 116), and acetylomithinase which hydrolyzes the N -acetylomithine to ornithine 116). It is highly significant that the last named enz3une was absent in extracts of a mutant blocked between iV -acetylomithine and ornithine 116). 

The reason for a double requirement of valine and isoleucine in certain mutant strains of Neurospora and E. colt is that a single enzyme catalyzes reactions common to both syntheses. Enz3mes for which there is such evidence are dihydroxy acid dehydrase (160) and a transaminase that converts a-ketovaline and a-ketoisoleucine to valine and isoleucine (143). There is a distinct probability that other enz3mes further back in the bios mthetic sequence also may function in common. 

Conversion of the a,j3-dihydroxy acids to the a-keto acids has been studied by Myers and Adelberg 169). These investigators observed that extracts of wild-type E. colt and Neurospora contain an enzyme(s) which dehydrates the dihydroxy acids. Mutants blocked at the conversion of the dihydroxy acids to the keto acids are deficient in this enzyme, and, also of a transaminase for the keto acids. 

Rudman and Meister IJ ) first showed the presence of a transaminase in cell-free extracts of E. colt that catalyze transamination reactions between glutamate and isoleucine, valine, leucine, norleucine, and norvaline. These monocarboxylic amino acids transaminated with each other as well as with glutamine. Preparations of an E. cdi mutant which did not respond to a-keto- 8-methylvalerate was unable to transaminate isoleucine or valine. The transaminase responsible for activity with the branched-chain amino acids was separated from other transaminases and considerably purified by standard methods of protein purification. It was shown to... 

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