Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity

Barber, Janet E B; Damry, Adam M.; Calderini, Guido F.; Walton, Curtis J. W.; Chica, Roberto A.

doi:10.1016/j.ab.2014.06.006

Cited by 17 publications

(15 citation statements)

References 25 publications

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“…To date, HRP is also a major component of high throughput assays in enzyme engineering, detecting H 2 O 2 as a side product of biooxidants (Willies et al 2012 ; Barber et al 2014 ) or products of coupling reactions after biohydroxylations (Joo et al 1999 ).…”

Section: Impact Of Recombinant Technology On Traditional and Future Hmentioning

confidence: 99%

An updated view on horseradish peroxidases: recombinant production and biotechnological applications

Krainer

Glieder

2015

Appl Microbiol Biotechnol

182

133

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Horseradish peroxidase has been the subject of scientific research for centuries. It has been used exhaustively as reporter enzyme in diagnostics and histochemistry and still plays a major role in these applications. Numerous studies have been conducted on the role of horseradish peroxidase in the plant and its catalytic mechanism. However, little progress has been made in its recombinant production. Until now, commercial preparations of horseradish peroxidase are still isolated from plant roots. These preparations are commonly mixtures of various isoenzymes of which only a small fraction has been described so far. The composition of isoenzymes in these mixed isolates is subjected to uncontrollable environmental conditions. Nowadays, horseradish peroxidase regains interest due to its broad applicability in the fields of medicine, life sciences, and biotechnology in cancer therapy, biosensor systems, bioremediation, and biocatalysis. These medically and commercially relevant applications, the recent discovery of new natural isoenzymes with different biochemical properties, as well as the challenges in recombinant production render this enzyme particularly interesting for future biotechnological solutions. Therefore, we reviewed previous studies as well as current developments with biotechnological emphasis on new applications and the major remaining biotechnological challenge—the efficient recombinant production of horseradish peroxidase enzymes.

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Section: Impact Of Recombinant Technology On Traditional and Future Hmentioning

confidence: 99%

An updated view on horseradish peroxidases: recombinant production and biotechnological applications

Krainer

Glieder

2015

Appl Microbiol Biotechnol

182

133

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“…We postulated that this lower activity with aromatic compounds is because the side chain of the d ‐amino acid substrate sits in a pocket formed by residues V33, S240, and T242 in the DAAT active site (Figure ) that is too small to bind the bulky benzyl side chain of d ‐phenylalanine efficiently. Previously, we demonstrated that replacement of V33 to a smaller glycine residue increased the catalytic efficiency ( k cat / K M ) of DAAT towards phenylpyruvate by over threefold, an enhancement that was mostly because of improved productive binding ( K M was decreased by approximately threefold). This result supports our hypothesis and suggests that mutations that increase the size of the side‐chain binding pocket in the active site will lead to the more efficient binding of bulkier substrates.…”

Section: Resultsmentioning

confidence: 88%

“…As expected, wild‐type DAAT displays a high specific activity with its native substrate d ‐alanine, reacts less efficiently with various aliphatic and polar substrates, and shows no detectable activity towards aromatic amino acids. Next, we screened the S240G and T242G mutants against the d ‐amino acid library, and included in this analysis the V33G variant that we described previously . We discovered that all three DAAT mutants display a broader substrate specificity than the wild‐type enzyme, and the V33G mutant has the broadest specificity as it reacts with all amino acids tested except for d ‐phenylglycine.…”

Section: Resultsmentioning

confidence: 99%

Engineered Aminotransferase for the Production of d‐Phenylalanine Derivatives Using Biocatalytic Cascades

et al. 2017

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d‐Phenylalanine derivatives are valuable chiral building blocks for a wide range of pharmaceuticals. Here, we developed stereoinversion and deracemization biocatalytic cascades to synthesize d‐phenylalanine derivatives that contain electron‐donating or ‐withdrawing substituents of various sizes and at different positions on the phenyl ring with a high enantiomeric excess (90 to >99 % ee) from commercially available racemic mixtures or l‐amino acids. These whole‐cell systems couple Proteus mirabilis l‐amino acid deaminase with an engineered aminotransferase that displays native‐like activity towards d‐phenylalanine, which we generated from Bacillus sp. YM‐1 d‐amino acid aminotransferase. Our cascades are applicable to preparative‐scale synthesis and do not require cofactor‐regeneration systems or chemical reducing agents.

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“…The activities of Pj AT, Cv AT, and Vf AT coupled with pyruvate amination were measured using an indirect assay by following alanine‐dependent reduction of NAD + by alanine dehydrogenase at 340 nm (ε NADH = 6.22 × 10 3 m −1 ·cm −1 ) . Excess alanine dehydrogenase from B. cereus ( Bc AlaDH) was added to the reaction mixtures to ensure that the rate‐limiting step is the transamination reaction.…”

Section: Methodsmentioning

confidence: 99%

Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism

et al. 2019

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The biodegradation of the nylon‐6 precursor caprolactam by a strain of Pseudomonas jessenii proceeds via ATP‐dependent hydrolytic ring opening to 6‐aminohexanoate. This non‐natural ω‐amino acid is converted to 6‐oxohexanoic acid by an aminotransferase (PjAT) belonging to the fold type I pyridoxal 5′‐phosphate (PLP) enzymes. To understand the structural basis of 6‐aminohexanoatate conversion, we solved different crystal structures and determined the substrate scope with a range of aliphatic and aromatic amines. Comparison with the homologous aminotransferases from Chromobacterium violaceum (CvAT) and Vibrio fluvialis (VfAT) showed that the PjAT enzyme has the lowest KM values (highest affinity) and highest specificity constant (kcat/KM) with the caprolactam degradation intermediates 6‐aminohexanoate and 6‐oxohexanoic acid, in accordance with its proposed in vivo function. Five distinct three‐dimensional structures of PjAT were solved by protein crystallography. The structure of the aldimine intermediate formed from 6‐aminohexanoate and the PLP cofactor revealed the presence of a narrow hydrophobic substrate‐binding tunnel leading to the cofactor and covered by a flexible arginine, which explains the high activity and selectivity of the PjAT with 6‐aminohexanoate. The results suggest that the degradation pathway for caprolactam has recruited an aminotransferase that is well adapted to 6‐aminohexanoate degradation. Database The atomic coordinates and structure factors P. jessenii 6‐aminohexanoate aminotransferase have been deposited in the PDB as entries http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4B (E∙succinate complex), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4C (E∙phosphate complex), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4D (E∙PLP complex), http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4E (E∙PLP‐6‐aminohexanoate intermediate), and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6G4F (E∙PMP complex).

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Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity

Cited by 17 publications

References 25 publications

An updated view on horseradish peroxidases: recombinant production and biotechnological applications

An updated view on horseradish peroxidases: recombinant production and biotechnological applications

Engineered Aminotransferase for the Production of d‐Phenylalanine Derivatives Using Biocatalytic Cascades

Biochemical properties of a Pseudomonas aminotransferase involved in caprolactam metabolism

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