scholarly journals Synthetic, structural and biological studies of the ubiquitin system: synthesis and crystal structure of an analogue containing unnatural amino acids

1997 ◽  
Vol 323 (3) ◽  
pp. 727-734 ◽  
Author(s):  
Steven G. LOVE ◽  
Tom W. MUIR ◽  
Robert RAMAGE ◽  
Kevin T. SHAW ◽  
Dmitriy ALEXEEV ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Side Chain ◽  
Hydrophobic Core ◽  
Biological Behaviour ◽  
Methyl Group ◽  
Ubiquitin System ◽  
Biological Studies ◽  
The Stability

Ubiquitin is a 76-amino acid protein involved in the targeting for destruction of proteins in the cell. The protein can readily be synthesized chemically affording an extra dimension to studies of protein stability. Ubiquitin with various modifications to the hydrophobic core has been synthesized. In particular, two core amino acids have been replaced by aminobutyric acid (Val-26) and norvaline (for Ile-30) and the product crystallized. The refined crystal structure shows an overall contraction of the molecule and the side chain of Nva-30 rotates relative to Ile-30. However, the side chain rotation is not sufficient to compensate for the effect of the loss of the methyl group and hence a small cavity is introduced into the structure, which decreases the stability of the protein. The biological behaviour of the modified protein is unaltered. The observed changes in stability are of the magnitude expected for the removal of methyl groups from the hydrophobic core of a protein. Interestingly, the effect appears to be independent of the position of the removed methyl group. The intact structure, but not its stability, is important for recognition by the biological conjugating system.

A Solid-Phase Synthetic Route to Unnatural Amino Acids with Diverse Side-Chain Substitutions

2002 ◽  
Vol 67 (9) ◽  
pp. 2960-2969 ◽  
Author(s):  
William L. Scott ◽  
Martin J. O'Donnell ◽  
Francisca Delgado ◽  
Jordi Alsina
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Solid Phase ◽  
Side Chain ◽  
Synthetic Route

A New and Efficient Synthesis of Unnatural Amino Acids and Peptides by Selective 3,3-Dimethyldioxirane Side-Chain Oxidation

1999 ◽  
Vol 64 (23) ◽  
pp. 8468-8474 ◽  
Author(s):  
Raffaele Saladino ◽  
Maurizio Mezzetti ◽  
Enrico Mincione ◽  
Ines Torrini ◽  
Mario Paglialunga Paradisi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Side Chain ◽  
Efficient Synthesis ◽  
Chain Oxidation

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

2019 ◽  
Author(s):  
Xinxin Zheng ◽  
Yinglu Cui ◽  
Tao Li ◽  
Ruifeng Li ◽  
Lu Guo ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Amino Acid ◽  
Unnatural Amino Acids ◽  
Catalytic Mechanism ◽  
Chiral Amines ◽  
Branched Chain Amino Acid ◽  
Chiral Amino Acids ◽  
Branched Chain ◽  
Substrate Spectrum

AbstractAminotransferases (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.

scholarly journals Crystal structure of HECT domain of UBE3C E3 ligase and its ubiquitination activity

2020 ◽  
Vol 477 (5) ◽  
pp. 905-923 ◽  
Author(s):  
Sunil Singh ◽  
J. Sivaraman
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Enzymatic Activity ◽  
Cancer Metastasis ◽  
E3 Ligase ◽  
The Other ◽  
Hect Domain ◽  
Loop Region ◽  
The Stability

The HECT family of E3 ubiquitin ligase is divided into three subfamilies: the NEDD4, the HERC, and the ‘other’. Previous studies have mostly targeted members of the NEDD4 subfamily for structural and functional analysis. The UBE3C E3 ligase is a member of the ‘other’ subfamily HECT and influences several crucial cellular processes, including innate immunity, proteasome processivity, and cancer metastasis. Here, we report the crystal structure of the HECT domain of UBE3C (amino acids (aa) 744–1083) with an additional fifty N-terminal amino acids (aa 693–743) at 2.7 Å, along with multiple in vitro ubiquitination assays to understand its enzymatic activity. The UBE3C HECT domain forms an open, L-shaped, bilobed conformation, having a large N-lobe and a small C-lobe. We show that the N-terminal region (aa 693–743) preceding the UBE3C HECT domain as well as a loop region (aa 758–762) in the N-lobe of the HECT domain affect the stability and activity of UBE3C HECT domain. Moreover, we identified Lys903 in the UBE3C HECT domain as a major site of autoubiquitination. The deletion of the last three amino acids at the C-terminal completely abrogated UBE3C activity while mutations of Gln961 and Ser1049 residues in the HECT domain substantially decreased its autoubiquitination activity. We demonstrate that these region/residues are involved in the E2–E3 transthiolation process and affect the UBE3C mediated autoubiquitination. Collectively, our study identified key residues crucial for UBE3C enzymatic activity, and it may assist in the development of suitable inhibitors to regulate its activity in multiple cancers.

Specificity of the transport system for neutral amino acids in the hamster intestine

1962 ◽  
Vol 202 (5) ◽  
pp. 919-925 ◽  
Author(s):  
Edmund C. C. Lin ◽  
Hiroshi Hagihira ◽  
T. Hastings Wilson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transport System ◽  
Side Chain ◽  
Carboxyl Group ◽  
Amino Group ◽  
Methyl Group ◽  
Neutral Amino Acids ◽  
Active Transport System ◽  
Everted Sacs

The specificity of the active transport system for neutral amino acids has been studied with everted sacs of hamster intestine. Amino acids with modifications or replacements of the carboxyl, amino, or α-hydrogen groups were poorly transported and were poor inhibitors of the transport of other l-amino acids. The carboxyl group must remain free, the amino group must not be in the tertiary or quaternary state, and the α-hydrogen can not be replaced by a methyl group without serious effect on the transport rate. It was concluded that the l-amino acids were distinguished from the d-isomers by the interaction of the carrier with the carboxyl group, the amino group, and the α-hydrogen. The side chain of the amino acid must be nonpolar but there is relatively little restriction on its structure.

Solid-phase synthesis and utilization of side-chain reactive unnatural amino acids

2003 ◽  
Vol 44 (46) ◽  
pp. 8403-8406 ◽  
Author(s):  
Martin J. O'Donnell ◽  
Jordi Alsina ◽  
William L. Scott
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
Side Chain ◽  
Phase Synthesis

scholarly journals The Effect of a Peptide Substrate Containing an Unnatural Branched Amino Acid on Chymotrypsin Activity

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 242
Author(s):  
Yuuki Yamawaki ◽  
Tomoki Yufu ◽  
Tamaki Kato
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Unnatural Amino Acids ◽  
Side Chain ◽  
Low Molecular Weight ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Reaction Velocity ◽  
Natural Amino Acids

7-Amino-4-methylcoumarin (AMC) is a low molecular weight fluorescent probe that can be attached to a peptide to enable the detection of specific proteases, such as chymotrypsin, expressed in certain diseases. Because this detection depends on the specificity of the protease toward the peptidyl AMC, the development of specific substrates is required. To investigate the specificity of chymotrypsin, peptidyl AMC compounds incorporating four different amino acid residues were prepared by liquid-phase synthesis. Two unnatural amino acids, 2-amino-4-ethylhexanoic acid (AEH) and cyclohexylalanine (Cha), were used to investigate the substrate specificity as these amino acids have structures different from natural amino acids. AEH was synthesized using diethyl acetamidemalonate as a starting material. The substrate containing Cha had high hydrophobicity and showed a high reaction velocity with chymotrypsin. Although the AEH substrate with a branched side chain had high hydrophobicity, it showed a low reaction velocity. The substrate containing the aromatic amino acid phenylalanine was less hydrophobic than the Cha and AEH substrates, but chymotrypsin showed the highest specificity for this compound. These results demonstrated that the substrate specificity of chymotrypsin is not only affected by the hydrophobicity and aromaticity, but also by the structural expanse of amino acid residues in the substrate.

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