| 1. |
- Engelmark Cassimjee, Karim, et al.
(författare)
-
A general protein purification and immobilization method on controlled porosity glass : biocatalytic applications
- 2014
-
Ingår i: Chemical Communications. - : Royal Society of Chemistry. - 1359-7345 .- 1364-548X. ; 50:65, s. 9134-9137
-
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.
|
|
| 2. |
- 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. - : Wiley. - 1742-464X .- 1742-4658. ; 279:5, s. 779-792
-
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).
|
|
| 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).
|
|
| 4. |
- Berglund, Per, et al.
(författare)
-
7.18 C-X Bond Formation : Transaminases as Chiral Catalysts: Mechanism, Engineering, and Applications
- 2012
-
Ingår i: Comprehensive Chirality. - : Elsevier. - 9780080951683 ; , s. 390-401
-
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.
|
|
| 5. |
- Cassimjee, Karim Engelmark, et al.
(författare)
-
Active Site Quantification of an omega-Transaminase by Performing a Half Transamination Reaction
- 2011
-
Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 1:9, s. 1051-1055
-
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).
|
|
| 6. |
- Cassimjee, Karim Engelmark, et al.
(författare)
-
Chromobacterium violaceum omega-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4 '-substituted acetophenones, and follows Swain-Lupton parameterisation
- 2012
-
Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 10:28, s. 5466-5470
-
Tidskriftsartikel (refereegranskat)abstract
- For biocatalytic production of pharmaceutically important chiral amines the.-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum omega-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (similar to 5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.
|
|
| 7. |
|
|
| 8. |
|
|
| 9. |
- Svedendahl Humble, Maria, et al.
(författare)
-
Biocatalytic Promiscuity
- 2011
-
Ingår i: European Journal of Organic Chemistry. - : John Wiley & Sons. - 1434-193X .- 1099-0690. ; :19, s. 3391-3401
-
Forskningsöversikt (refereegranskat)abstract
- Enzymes are attractive catalysts because of their promiscuity and their ability to perform highly regio-, chemo- and stereo-selective transformations. Enzyme promiscuity allows optimisation of industrial processes that require reaction conditions different from those in nature. Many enzymes can be used in reactions completely different from the reaction the enzyme originally evolved to perform. Such catalytically promiscuous reactions can be secondary activities hidden behind a native activity and might be discovered either in screening for that particular activity or, alternatively, by chance. Recently, researchers have designed enzymes to show catalytic promiscuity. It is also possible to design new enzymes from scratch by computer modelling (de novo design), but most work published to date starts from a known enzyme backbone. Promiscuous activity might also be induced or enhanced by rational design or directed evolution (or combinations thereof). Enzyme catalytic promiscuity provides fundamental knowledge about enzyme/substrate interactions and the evolution of new enzymes. New enzymes are required by industry, which needs to optimise chemical processes in an environmentally sustainable way. In this review various aspects of enzyme catalytic promiscuity are considered from a biocatalytic perspective.
|
|
| 10. |
- Svedendahl Humble, Maria, et al.
(författare)
-
Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an omega-Transaminase
- 2012
-
Ingår i: ChemCatChem. - : Wiley. - 1867-3880 .- 1867-3899. ; 4:8, s. 1167-1172
-
Tidskriftsartikel (refereegranskat)abstract
- Transaminases inherently possess high enantiospecificity and are valuable tools for stereoselective synthesis of chiral amines in high yield from a ketone and a simple amino donor such as 2-propylamine. Most known ?-transaminases are (S)-selective and there is, therefore, a need of (R)-selective enzymes. We report the successful rational design of an (S)-selective ?-transaminase for reversed and improved enantioselectivity. Previously, engineering performed on this enzyme group was mainly based on directed evolution, with few exceptions. One reason for this is the current lack of 3D structures. We have explored the ?-transaminase from Chromobacterium violaceum and have used a homology modeling/rational design approach to create enzyme variants for which the activity was increased and the enantioselectivity reversed. This work led to the identification of key amino acid residues that control the activity and enantiomeric preference. To increase the enantiospecificity of the C. violaceum ?-transaminase, a possible single point mutation (W60C) in the active site was identified by homology modeling. By site-directed mutagenesis this enzyme variant was created and it displayed an E value improved up to 15-fold. In addition, to reverse the enantiomeric preference of the enzyme, two other point mutations (F88A/A231F) were identified. This double mutation created an enzyme variant, which displayed substrate dependent reversed enantiomeric preference with an E value shifted from 3.9 (S) to 63 (R) for 2-aminotetralin.
|
|
