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Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.
KTH, Skolan för bioteknologi (BIO), Biokemi.ORCID-id: 0000-0003-2371-8755
KTH, Skolan för bioteknologi (BIO), Biokemi.
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2012 (Engelska)Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, nr 5, s. 779-792Artikel i tidskrift (Refereegranskat) 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).

Ort, förlag, år, upplaga, sidor
2012. Vol. 279, nr 5, s. 779-792
Nationell ämneskategori
Strukturbiologi
Identifikatorer
URN: urn:nbn:se:kth:diva-74820DOI: 10.1111/j.1742-4658.2012.08468.xISI: 000300665900009PubMedID: 22268978Scopus ID: 2-s2.0-84857650697OAI: oai:DiVA.org:kth-74820DiVA, id: diva2:490047
Anmärkning
QC 20120309Tillgänglig från: 2012-02-03 Skapad: 2012-02-03 Senast uppdaterad: 2017-12-08Bibliografiskt granskad
Ingår i avhandling
1. ω-Transaminase in Biocatalysis: Methods, Reactions and Engineering
Öppna denna publikation i ny flik eller fönster >>ω-Transaminase in Biocatalysis: Methods, Reactions and Engineering
2012 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2012. s. x, 57
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2012:13
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-92516 (URN)978-91-7501-242-1 (ISBN)
Disputation
2012-04-20, FR4 (Oscar Kleins Auditorium) AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning
QC 20120402Tillgänglig från: 2012-04-02 Skapad: 2012-04-02 Senast uppdaterad: 2012-04-02Bibliografiskt granskad

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