scholarly journals Editing of non-cognate aminoacyl adenylates by peptide synthetases

1999 ◽  
Vol 342 (3) ◽  
pp. 715-719 ◽  
Author(s):  
Maja PAVELA-VRANCIC ◽  
Ralf DIECKMANN ◽  
Hans VON DÖHREN ◽  
Horst KLEINKAUF
Keyword(s):  
Amino Acid ◽  
Aromatic Amino Acids ◽  
Substrate Selection ◽  
Peptide Synthetases ◽  
Substrate Analogues ◽  
Wide Range ◽  
The Stability ◽  
Acid Substrate ◽  
Amino Acid Substrate ◽  
Selection Of

Non-ribosomally formed peptides display both highly conserved and variable amino acid positions, the variations leading to a wide range of peptide families. Activation of the amino acid substrate proceeds in analogy to the ribosomal biosynthetic mechanism generating aminoacyl adenylate and acyl intermediates. To approach the mechanism of fidelity of amino acid selection, the stability of the aminoacyl adenylates was studied by employing a continuous coupled spectrophotometric assay. The apo-form of tyrocidine synthetase 1 (apo-TY1) was used, generating an L-phenylalanyl-adenylate intermediate stabilized by the interaction of two structural subdomains of the adenylation domain. Adenylates of substrate analogues have shown variable and reduced degrees of stability, thus leading to an enhanced generation of pyrophosphate due to hydrolysis and continuous adenylate formation. Discrimination of the non-aromatic amino acids L-Leu and L-Met, or L-Phe analogues such as p-amino- and p-chloro-L-Phe derivatives, as well as the stereospecific selection of L-Phe, is supported by less-stable adenylate intermediates exhibiting elevated susceptibility to hydrolysis. Breakdown of the L-phenylalanyl intermediate utilizing 2′-deoxy-ATP as the nucleotide substrate was significantly enhanced compared with the natural analogue. Apo-TY1 engineered at positions involved in adenylate formation showed variable protection against hydrolysis. The results imply that stability of the aminoacyl intermediates may act as an essential factor in substrate selection and fidelity of non-ribosomal-peptide-forming systems.

ATP Binding Plays a Role in the Selection of Amino Acid Substrate by Aminoacyl-tRNA Synthetases

1990 ◽  
Vol 613 (1 Enzyme Engine) ◽  
pp. 489-493 ◽  
Author(s):  
BEN'ICHIRO TONOMURA ◽  
MAKOTO KAKITANI ◽  
YUJI OHKUBO ◽  
HIDEAKI SHIMA ◽  
KEITARO HIROMI
Keyword(s):  
Amino Acid ◽  
Atp Binding ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Acid Substrate ◽  
Amino Acid Substrate ◽  
Selection Of

scholarly journals Use of β3-methionine as an amino acid substrate of Escherichia coli methionyl-tRNA synthetase

2020 ◽  
Vol 209 (2) ◽  
pp. 107435 ◽  
Author(s):  
Giuliano Nigro ◽  
Sophie Bourcier ◽  
Christine Lazennec-Schurdevin ◽  
Emmanuelle Schmitt ◽  
Philippe Marlière ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Trna Synthetase ◽  
Methionyl Trna Synthetase ◽  
Acid Substrate ◽  
Amino Acid Substrate

scholarly journals Reprogramming Versus Subdomain Substitution: An Exploration of Strategies for NRPS Engineering Using the Unique Model System BpsA

2021 ◽  
Author(s):  
◽  
Vincent Collins
Keyword(s):  
Assembly Line ◽  
Binding Pocket ◽  
Model System ◽  
Blue Pigment ◽  
Adenylation Domain ◽  
Dynamic Nature ◽  
Peptide Synthetases ◽  
Acid Substrate ◽  
Existing Structure ◽  
Amino Acid Substrate

<p>Non-ribosomal peptide synthetases (NRPSs) are large enzymes that generate a plethora of important natural products, from antibiotics to immunosuppressants. These modular enzymes function like an assembly line, selecting and incorporating specific (and frequently nonproteinogenic) amino acids into a growing peptide chain. This modular structure offers promise for re-engineering NRPS units to generate new useful products, but progress has to date been limited by the complex and dynamic nature of key domains, and a failure to define generally applicable “rules” to guide engineering efforts. Early efforts to engineer NRPS enzymes relied on the substitution of entire NRPS modules or domains, but product yields were often very low. However, these studies did highlight the promise of targeting the adenylation domain, the part of each NRPS modules that is responsible for selecting each amino acid substrate. Two particularly promising strategies for NRPS engineering aim to manipulate the adenylation domain in ways that minimise steric disruption to the assembly line. The first of these, reprogramming, makes the fewest possible changes to the NRPS primary sequence, but is dependent on those precise changes conforming to the existing structure of the adenylation domain binding pocket. More recently a second technique has been developed, subdomain substitution, which recombines a larger region of the adenylation domain to avoid perturbation of the binding pocket. The research described in this thesis examined and compared both approaches using the unique NRPS BpsA as a model system. BpsA is a single-module NRPS that generates a vivid blue pigment product, making for a reductionist system that offers a robust visual reporter capacity. Experiments with the reprogramming technique showed that small changes to the protein sequence had potential to exert major impacts on enzyme function, even when no change to function was intended. In contrast, experiments with subdomain substitution were generally more effective, showing that NRPS enzymes are very sensitive to the precise boundaries of the substituted region, but that activity can be restored to otherwise non-functional subdomain substitutions by modulation of the regional boundaries.</p>

scholarly journals Tyrosine Aminotransferase Catalyzes the Final Step of Methionine Recycling in Klebsiella pneumoniae

1999 ◽  
Vol 181 (6) ◽  
pp. 1739-1747 ◽  
Author(s):  
Jacqueline Heilbronn ◽  
Judith Wilson ◽  
Bradley J. Berger
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Amino Acid Sequence ◽  
Paracoccus Denitrificans ◽  
Rhizobium Meliloti ◽  
Aromatic Amino Acids ◽  
Tyrosine Aminotransferase ◽  
E Coli ◽  
Substrate Analogues ◽  
Terminal Amino

ABSTRACT An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of α-ketomethiobutyrate to methionine, has been purified fromKlebsiella pneumoniae and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products). The structural gene for the tyrosine aminotransferase was cloned from K. pneumoniae and expressed in E. coli. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E. coli, S. typhimurium, Paracoccus denitrificans, andRhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the K. pneumoniae enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with α-ketomethiobutyrate and of glutamate with α-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with α-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.

Human 20S proteasome activity towards fluorogenic peptides of various chain lengths

2016 ◽  
Vol 397 (9) ◽  
pp. 921-926 ◽  
Author(s):  
Wioletta Rut ◽  
Marcin Drag
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Kinetic Analysis ◽  
20S Proteasome ◽  
Fluorogenic Substrates ◽  
Catalytic Activities ◽  
Peptide Length ◽  
Chain Lengths ◽  
Acid Substrate ◽  
Amino Acid Substrate

Abstract The proteasome is a multicatalytic protease responsible for the degradation of misfolded proteins. We have synthesized fluorogenic substrates in which the peptide chain was systematically elongated from two to six amino acids and evaluated the effect of peptide length on all three catalytic activities of human 20S proteasome. In the cases of five- and six-membered peptides, we have also synthesized libraries of fluorogenic substrates. Kinetic analysis revealed that six-amino-acid substrates are significantly better for chymotrypsin-like and caspase-like activity than shorter peptidic substrates. In the case of trypsin-like activity, a five-amino-acid substrate was optimal.

The amino acid substrate of bovine tyrosine hydroxylase

1992 ◽  
Vol 21 (2) ◽  
pp. 191-196 ◽  
Author(s):  
Marc M. Meyer ◽  
Paul F. Fitzpatrick
Keyword(s):  
Amino Acid ◽  
Tyrosine Hydroxylase ◽  
Acid Substrate ◽  
Amino Acid Substrate

scholarly journals The existence of an insulin-stimulated glucose and non-essential but not essential amino acid substrate interaction in diabetic pigs

2011 ◽  
Vol 12 (1) ◽  
pp. 25 ◽  
Author(s):  
Sietse J Koopmans ◽  
Jan VanderMeulen ◽  
Jan Wijdenes ◽  
Henk Corbijn ◽  
Ruud Dekker
Keyword(s):  
Amino Acid ◽  
Essential Amino Acid ◽  
Substrate Interaction ◽  
Acid Substrate ◽  
Amino Acid Substrate
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