Cloning and characterization of a novel fold-type I branched-chain amino acid aminotransferase from the hyperthermophilic archaeon Thermococcus sp. CKU-1

Uchida, Yuki; Hayashi, Hideyuki; Washio, Tsubasa; Yamasaki, Ryo; Kato, Shiro; Oikawa, Tadao

doi:10.1007/s00792-014-0642-0

Cited by 20 publications

(7 citation statements)

References 43 publications

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“…In amino acid utilizing enzymes other than AARSs, the highest selectivity against smaller hydrophobic amino acids that we could identify was for a branched amino acid aminotransferase whose preferred substrate is Ile. Its discrimination factor for Val is 9.2 and for Abu 34.5 (Table S1) [126], compared to IleRS with discrimination factors of 150 and 7100 for Val and Abu, respectively (Table 1) [27,34]. Further, even when selectivity is examined in a wider context, that is, for any enzyme with a noncognate substrate which is one methylene group smaller than the cognate one, AARSs lead the selectivity record (a recent analysis indicated a selectivity ceiling for CH 3 versus H of 160; fig.…”

Section: The Selectivity Of Other Amino Acid Utilizing Enzymesmentioning

confidence: 99%

How evolution shapes enzyme selectivity – lessons from aminoacyl‐tRNA synthetases and other amino acid utilizing enzymes

Tawfik

Gruić-Sovulj

2020

The FEBS Journal

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Aminoacyl‐tRNA synthetases (AARSs) charge tRNA with their cognate amino acids. Many other enzymes use amino acids as substrates, yet discrimination against noncognate amino acids that threaten the accuracy of protein translation is a hallmark of AARSs. Comparing AARSs to these other enzymes allowed us to recognize patterns in molecular recognition and strategies used by evolution for exercising selectivity. Overall, AARSs are 2–3 orders of magnitude more selective than most other amino acid utilizing enzymes. AARSs also reveal the physicochemical limits of molecular discrimination. For example, amino acids smaller by a single methyl moiety present a discrimination ceiling of ~200, while larger ones can be discriminated by up to 105‐fold. In contrast, substrates larger by a hydroxyl group challenge AARS selectivity, due to promiscuous H‐bonding with polar active site groups. This ‘hydroxyl paradox’ is resolved by editing. Indeed, when the physicochemical discrimination limits are reached, post‐transfer editing – hydrolysis of tRNAs charged with noncognate amino acids, evolved. The editing site often selectively recognizes the edited noncognate substrate using the very same feature that the synthetic site could not efficiently discriminate against. Finally, the comparison to other enzymes also reveals that the selectivity of AARSs is an explicitly evolved trait, showing some clear examples of how selection acted not only to optimize catalytic efficiency with the target substrate, but also to abolish activity with noncognate threat substrates (‘negative selection’).

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Section: The Selectivity Of Other Amino Acid Utilizing Enzymesmentioning

confidence: 99%

How evolution shapes enzyme selectivity – lessons from aminoacyl‐tRNA synthetases and other amino acid utilizing enzymes

Tawfik

Gruić-Sovulj

2020

The FEBS Journal

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“…Branched chain TAms (BCATs) catalyse reversible transamination of branched chain amino acids (shown in Scheme 1). Recently, archaeal thermophilic BCATs have been biochemically characterized from Thermococcus sp (Uchida et al, 2014) and biochemically and structurally studied from the thermophile Thermoproteus uzoniensis (Boyko et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Thermostable Branched-Chain Amino Acid Transaminases From the Archaea Geoglobus acetivorans and Archaeoglobus fulgidus: Biochemical and Structural Characterization

Isupov

Boyko

Sutter

et al. 2019

Front. Bioeng. Biotechnol.

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Two new thermophilic branched chain amino acid transaminases have been identified within the genomes of different hyper-thermophilic archaea, Geoglobus acetivorans, and Archaeoglobus fulgidus. These enzymes belong to the class IV of transaminases as defined by their structural fold. The enzymes have been cloned and over-expressed in Escherichia coli and the recombinant enzymes have been characterized both biochemically and structurally. Both enzymes showed high thermostability with optimal temperature for activity at 80 and 85°C, respectively. They retain good activity after exposure to 50% of the organic solvents, ethanol, methanol, DMSO and acetonitrile. The enzymes show a low activity to (R)-methylbenzylamine but no activity to (S)-methylbenzylamine. Both enzymes have been crystallized and their structures solved in the internal aldimine form, to 1.9 Å resolution for the Geoglobus enzyme and 2.0 Å for the Archaeoglobus enzyme. Also the Geoglobus enzyme structure has been determined in complex with the amino acceptor α-ketoglutarate and the Archaeoglobus enzyme in complex with the inhibitor gabaculine. These two complexes have helped to determine the conformation of the enzymes during enzymatic turnover and have increased understanding of their substrate specificity. A comparison has been made with another (R) selective class IV transaminase from the fungus Nectria haematococca which was previously studied in complex with gabaculine. The subtle structural differences between these enzymes has provided insight regarding their different substrate specificities.

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“…However, the temperature preference of P sBCAT exhibited significant differences compared with thermal BCATs from Thermoproteus uzoniensis (35, 36), Thermococcus sp. CKU-1 (37) and Vulcanisaeta moutnovskia (34), which showed maximum activity at > 90 °C.…”

Section: Resultsmentioning

confidence: 99%

Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

Zheng

Cui

et al. 2019

Preprint

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Aminotransferases (ATs) are important biocatalysts for the synthesis of chiral amines because of their capability of introducing amino group into ketones or keto acids as well as their high enantioselectivity, high regioselectivity and no requirement of external addition of cofactor. Among all ATs, branched-chain amino acid aminotransferase (BCAT) can reversibly catalyse branched-chain amino acids (BCAAs), including L-valine, L-leucine, and L-isoleucine, with α-ketoglutaric acid to form the corresponding ketonic acids and L-glutamic acid. Alternatively, BCATs have been used for the biosynthesis of unnatural amino acids, such as L-tert-leucine. In the present study, the BCAT from Pseudomonas sp. (PsBCAT) was cloned and expressed in Escherichia coli for biochemical and structural analyses. The optimal reaction temperature and pH of PsBCAT were 40 °C and 8.5, respectively. PsBCAT exhibited a comparatively broader substrate spectrum, and showed remarkably high activity with L-leucine, L-valine, L-isoleucine and L-methionine with activities of 105 U/mg, 127 U/mg, 115 U/mg and 98 U/mg, respectively. Additionally, PsBCAT had activities with aromatic L-amino acids, L-histidine, L-lysine, and L-threonine. To analyse the catalytic mechanism of PsBCAT with the broad substrate spectrum, the crystal structure of PsBCAT was also determined. Finally, conjugated with the ornithine aminotransferase (OrnAT) from Bacillus subtilis, the coupled system was applied to the preparation of L-tert-leucine with 83% conversion, which provided an approximately 2.7-fold higher yield than the single BCAT reaction.IMPORTANCEDespite the enormous potential of BCATs, the vast majority of enzymes still lack suitably broad substrate scope and activity, thus new sources and novel enzymes are currently being investigated. Here, we described a previously uncharacterized PsBCAT, which showed a surprisingly wide substrate range and was more active towards BCAAs. This substrate promiscuity is unique for the BCAT family and could prove useful in industrial applications. Based on the determined crystal structure, we found some differences in the organization of the substrate binding cavity, which may influence the substrate specificity of the enzyme. Moreover, we demonstrated efficient biocatalytic asymmetric synthesis of L-tert-leucine using a coupling system, which can be used to remove the inhibitory by-product, and to shift the reaction equilibrium towards the product formation. In summary, the structural and functional characteristics of PsBCAT were analysed in detail, and this information will play an important role in the synthesis of chiral amino acids and will be conducive to industrial production of enantiopure chiral amines by aminotransferase.

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Cloning and characterization of a novel fold-type I branched-chain amino acid aminotransferase from the hyperthermophilic archaeon Thermococcus sp. CKU-1

Cited by 20 publications

References 43 publications

How evolution shapes enzyme selectivity – lessons from aminoacyl‐tRNA synthetases and other amino acid utilizing enzymes

How evolution shapes enzyme selectivity – lessons from aminoacyl‐tRNA synthetases and other amino acid utilizing enzymes

Thermostable Branched-Chain Amino Acid Transaminases From the Archaea Geoglobus acetivorans and Archaeoglobus fulgidus: Biochemical and Structural Characterization

Biochemical and structural characterization of a highly active branched-chain amino acid aminotransferase from Pseudomonas sp. for efficient biosynthesis of chiral amino acids

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