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Key Amino Acid Residues for Reversed or Improved Enantiospecificity of an omega-Transaminase
KTH, School of Biotechnology (BIO), Biochemistry.ORCID iD: 0000-0003-2371-8755
KTH, School of Biotechnology (BIO), Biochemistry.
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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.

Place, publisher, year, edition, pages
2012. Vol. 4, no 8, 1167-1172 p.
Keyword [en]
Amination, Biocatalysis, Enantiopreference, Enantiospecificity, Protein engineering
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-92286DOI: 10.1002/cctc.201100487ISI: 000306907700020Scopus ID: 2-s2.0-84864374814OAI: oai:DiVA.org:kth-92286DiVA: diva2:513150
Note

QC 20120905

Available from: 2012-03-30 Created: 2012-03-30 Last updated: 2017-12-07Bibliographically approved
In thesis
1. ω-Transaminase in Biocatalysis: Methods, Reactions and Engineering
Open this publication in new window or tab >>ω-Transaminase in Biocatalysis: Methods, Reactions and Engineering
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:nbn:se:kth:diva-92516 (URN)978-91-7501-242-1 (ISBN)
Public defence
2012-04-20, FR4 (Oscar Kleins Auditorium) AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (English)
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Note
QC 20120402Available from: 2012-04-02 Created: 2012-04-02 Last updated: 2012-04-02Bibliographically approved

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Svedendahl Humble, MariaBerglund, Per

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