| 1. |
- Engelmark Cassimjee, Karim, et al.
(författare)
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A general protein purification and immobilization method on controlled porosity glass : biocatalytic applications
- 2014
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Ingår i: Chemical Communications. - : Royal Society of Chemistry. - 1359-7345 .- 1364-548X. ; 50:65, s. 9134-9137
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Tidskriftsartikel (refereegranskat)abstract
- A general combined purification and immobilization method to facilitate biocatalytic process development is presented. The support material, EziG (TM), is based on controlled porosity glass (CPG) or polymer-coated versions thereof (HybCPG) and binds protein affinity tags. Biocatalytic reactions in aqueous and organic media with seven enzymes of biocatalytic interest are shown.
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| 2. |
- Svedendahl Humble, Maria, et al.
(författare)
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Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.
- 2012
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 279:5, s. 779-792
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Tidskriftsartikel (refereegranskat)abstract
- The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC2.6.1.18) catalyses industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular mass of ∼ 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique 'relaxed' conformations of three critical loops involved in structuring the active site that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered 'roof ' over the PLP-binding site, whereas in the other apo form and the holo form the 'roof' is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the 'roof' structure may be associated with substrate binding in Cv-ωTA and some other transaminases. DATABASE: The atomic coordinates and structure factors for the Chromobacterium violaceumω-transaminase crystal structures can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession codes 4A6U for the holoenzyme, 4A6R for the apo1 form, 4A6T for the apo2 form and 4A72 for the mixed form STRUCTURED DIGITAL ABSTRACT: • -transaminases and -transaminases bind by dynamic light scattering (View interaction) • -transaminase and -transaminase bind by x-ray crystallography (View interaction) • -transaminase and -transaminase bind by x-ray crystallography (View interaction).
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| 3. |
- Svedendahl Humble, Maria, et al.
(författare)
-
Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.
- 2012
-
Ingår i: The FEBS Journal. - 1742-464X. ; 279:5, s. 779-792
-
Tidskriftsartikel (refereegranskat)abstract
- SUMMARY: The bacterial ω-transaminase from Chromobacterium violaceum (Cv-ωTA, EC 2.6.1.18) catalyzes industrially important transamination reactions by use of the coenzyme pyridoxal 5'-phosphate (PLP). Here, we present four crystal structures of Cv-ωTA: two in the apo form, one in the holo form and one in an intermediate state, at resolutions between 1.35 and 2.4 Å. The enzyme is a homodimer with a molecular weight of approximately 100 kDa. Each monomer has an active site at the dimeric interface that involves amino acid residues from both subunits. The apo-Cv-ωTA structure reveals unique "relaxed" conformations of three critical loops involved in structuring the active site, that have not previously been seen in a transaminase. Analysis of the four crystal structures reveals major structural rearrangements involving elements of the large and small domains of both monomers that reorganize the active site in the presence of PLP. The conformational change appears to be triggered by binding of the phosphate group of PLP. Furthermore, one of the apo structures shows a disordered "roof" over the PLP binding site, while in the other apo form and the holo form the "roof" is ordered. Comparison with other known transaminase crystal structures suggests that ordering of the "roof" structure may be associated with substrate binding in Cv-ωTA and some other transaminases. STRUCTURED DIGITAL ABSTRACT: -transaminases and -transaminases bind by dynamic light scattering (View interaction) -transaminase and -transaminase bind by x-ray crystallography (View interaction) -transaminase and -transaminase bind by x-ray crystallography (View interaction).
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| 4. |
- Berglund, Per, et al.
(författare)
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7.18 C-X Bond Formation : Transaminases as Chiral Catalysts: Mechanism, Engineering, and Applications
- 2012
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Ingår i: Comprehensive Chirality. - : Elsevier. - 9780080951683 ; , s. 390-401
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Bokkapitel (refereegranskat)abstract
- Enantiomerically pure amines and amino acids are important building blocks in academic research as well as in industrial-scale chemical production. Transaminases are versatile enzymes providing access to such compounds of high enantiomeric excess. This chapter illustrates the available strategies with transaminases such as kinetic resolution or stereoselective synthesis and highlights many successful examples for amino acid and chiral amines synthesis. There are some known challenges linked to the use of transaminases, for example in terms of unfavorable equilibria and inhibition. Several successful examples to overcome these limitations are presented. Also, the classification of transaminases, mechanistic details, and various strategies for optimization are discussed.
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| 5. |
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| 6. |
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| 7. |
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| 8. |
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| 9. |
- Carlqvist, Peter, et al.
(författare)
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Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
- 2005
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 6, s. 331-336
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Tidskriftsartikel (refereegranskat)abstract
- Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant locks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular-dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The k(cat) values were found to be in the range of 10(-3) to 4 min(-1), similar to the values obtained with aldolase antibodies. The enzyme proficiency was 10(7). Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.
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| 10. |
- Cassimjee, Karim Engelmark, et al.
(författare)
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Active Site Quantification of an omega-Transaminase by Performing a Half Transamination Reaction
- 2011
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 1:9, s. 1051-1055
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Tidskriftsartikel (refereegranskat)abstract
- Measurement of the active enzyme fraction in a given enzyme preparation is a requirement for accurate kinetic measurements and activity comparisons of, for example, engineered mutants. omega-Transaminases, enzymes capable of interconverting ketones and amines by use of pyridoxal-5'-phosphate (PIP), can be used for the production of pharmaceutically important chiral amines but are subject to engineering to meet the practical requirements in synthesis reactions. Therefore, an active site quantification method is needed. Such a method was developed by quantifying the amount of consumed substrate in a virtually irreversible half transamination reaction. (S)-1-phenylethylamine was converted to acetophenone, while the holo enzyme (E-PLP) was converted to apo enzyme with bound pyridoxamine-5'-phosphate (E:PMP). Further, the mass of active enzyme was correlated to the absorbance of the holo enzyme to achieve a direct measurement method. The active Chromobacterium violaceum omega-transaminase with bound PLP can be quantified at 395 nm with an apparent extinction coefficient of 8.1 mM(-1) cm(-1).
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