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ω-Transaminase in Biocatalysis: Methods, Reactions and Engineering
KTH, School of Biotechnology (BIO), Biochemistry.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biocatalysis offers an alternative to classic chemistry by using enzymes, the protein catalysts of Nature, for production of fine chemicals. Evolution has created enzymes capable of catalysis at moderate temperature of a specific reaction in the presence of a plethora of compounds in the aqueous cell environment. The focal point of biocatalysis is to utilise these traits in vitro, for creation of valuable molecules.

The ω-transaminase is an enzyme capable of producing chiral amines, compounds used to great extent in pharmaceuticals. Much effort has in recent years been invested in the research and engineering of this enzyme type since the catalysed reaction offers an advantageous alternative to classical techniques. Nevertheless, there is a need for method development, adaptation of the enzyme and increased understanding of the catalytic mechanism for feasibility as an effective biocatalyst for unnatural substrates. This thesis addresses a chosen set of obstacles as a contribution to meeting the demands at hand. ω-Transaminase from Chromobacterium violaceum and Arthrobacter citreus was used.

Many homologous ω-transaminases are available, which are also subject to engineering where variants are produced. To accurately compare their kinetic constants an active site quantification method is required but has not been available. Here such a method is presented (Paper 1) which encompasses a virtually irreversible half transamination reaction.

In stereoselective synthesis the ω-transaminase catalysed equilibrium reaction inherently results in incomplete conversion. An equilibrium displacement system is presented (Paper II) where isopropylamine is the amino donor for transamination of acetophenone and derivatives thereof, coupled to an enzymatic cascade reaction.

For many unnatural substrates the specificity and enantiospecificity is insufficient. Rationally redesigned variants were produced with improved properties for chosen substrates (Paper III and IV). The catalytic contributions of field and resonance of a variant compared to the wild type were investigated (Paper IV) for increased knowledge of the mechanism.

For rational redesign of an enzyme the three-dimensional structure is required, of which only a few are available for the ω-transaminases. X-ray crystallographic structures of the holo and apo form of Chromobacterium violaceum ω-transaminase were made (Paper V) which revealed significant structural rearrangements upon coenzyme binding which may be of consequence for future engineering.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. , x, 57 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2012:13
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-92516ISBN: 978-91-7501-242-1 (print)OAI: oai:DiVA.org:kth-92516DiVA: diva2:513329
Public defence
2012-04-20, FR4 (Oscar Kleins Auditorium) AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20120402Available from: 2012-04-02 Created: 2012-04-02 Last updated: 2012-04-02Bibliographically approved
List of papers
1. Active Site Quantification of an omega-Transaminase by Performing a Half Transamination Reaction
Open this publication in new window or tab >>Active Site Quantification of an omega-Transaminase by Performing a Half Transamination Reaction
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2011 (English)In: ACS CATAL, ISSN 2155-5435, Vol. 1, no 9, 1051-1055 p.Article in journal (Refereed) Published
Abstract [en]

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).

Keyword
aminotransferase, chiral amines, pyridoxal-5 '-phosphate, PLP, biocatalysis, enzyme kinetics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-41299 (URN)10.1021/cs200315h (DOI)000294704500010 ()
Note
QC 20110927Available from: 2011-09-27 Created: 2011-09-26 Last updated: 2012-04-02Bibliographically approved
2. Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration
Open this publication in new window or tab >>Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration
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2010 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 46, no 30, 5569-5571 p.Article in journal (Refereed) Published
Abstract [en]

Enantiopure chiral amines synthesis using omega-transaminases is hindered by an unfavourable equilibrium, but when using isopropylamine as the amine donor the equilibrium can be completely displaced by using a specific dehydrogenase in situ for removal of formed acetone.

Keyword
OPTICALLY-ACTIVE AMINES, ASYMMETRIC-SYNTHESIS, OMEGA-TRANSAMINASES, CHIRAL AMINES
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-12935 (URN)10.1039/c0cc00050g (DOI)000280145000048 ()
Note
QC 20100519Available from: 2010-05-19 Created: 2010-05-19 Last updated: 2017-12-12Bibliographically approved
3. Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an omega-Transaminase
Open this publication in new window or tab >>Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an omega-Transaminase
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2012 (English)In: ChemCatChem, ISSN 1867-3880, E-ISSN 1867-3899, Vol. 4, no 8, 1167-1172 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Amination, Biocatalysis, Enantiopreference, Enantiospecificity, Protein engineering
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-92286 (URN)10.1002/cctc.201100487 (DOI)000306907700020 ()2-s2.0-84864374814 (Scopus ID)
Note

QC 20120905

Available from: 2012-03-30 Created: 2012-03-30 Last updated: 2017-12-07Bibliographically approved
4. Chromobacterium violaceum ω-Transaminase VariantTrp60Cys Shows Increased Specificity for (S)-1-Phenylethylamine and 4’-Substituted Acetophenones, andFollows Swain-Lupton Parameterisation
Open this publication in new window or tab >>Chromobacterium violaceum ω-Transaminase VariantTrp60Cys Shows Increased Specificity for (S)-1-Phenylethylamine and 4’-Substituted Acetophenones, andFollows Swain-Lupton Parameterisation
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-92322 (URN)
Note
QS 2012Available from: 2012-04-02 Created: 2012-04-02 Last updated: 2012-04-02Bibliographically approved
5. Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.
Open this publication in new window or tab >>Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.
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2012 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 5, 779-792 p.Article in journal (Refereed) Published
Abstract [en]

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).

National Category
Structural Biology
Identifiers
urn:nbn:se:kth:diva-74820 (URN)10.1111/j.1742-4658.2012.08468.x (DOI)000300665900009 ()22268978 (PubMedID)2-s2.0-84857650697 (Scopus ID)
Note
QC 20120309Available from: 2012-02-03 Created: 2012-02-03 Last updated: 2017-12-08Bibliographically approved

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