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FEBS J
2014 May 01;2819:2240-53. doi: 10.1111/febs.12778.
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The substrate specificity, enantioselectivity and structure of the (R)-selective amine : pyruvate transaminase from Nectria haematococca.
Sayer C
,
Martinez-Torres RJ
,
Richter N
,
Isupov MN
,
Hailes HC
,
Littlechild JA
,
Ward JM
.
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UNLABELLED: During the last decade the use of transaminases for the production of pharmaceutical and fine chemical intermediates has attracted a great deal of attention. Transaminases are versatile biocatalysts for the efficient production of amine intermediates and many have (S)-enantiospecificity. Transaminases with (R)-specificity are needed to expand the applications of these enzymes in biocatalysis. In this work we have identified a fungal putative (R)-specific transaminase from the Eurotiomycetes Nectria haematococca, cloned a synthetic version of this gene, demonstrated (R)-selective deamination of several substrates including (R)-α-methylbenzylamine, as well as production of (R)-amines, and determined its crystal structure. The crystal structures of the holoenzyme and the complex with an inhibitor gabaculine offer the first detailed insight into the structural basis for substrate specificity and enantioselectivity of the industrially important class of (R)-selective amine : pyruvate transaminases.
DATABASE: The atomic coordinates and structure factors for the Nectria TAm in holoenzyme and gabaculine-bound forms have been deposited in the PDB as entries 4cmd and 4cmf respectively.
Figure 6. SchemeA representation of the transaminase enzyme mechanism showing key intermediates.
Figure 1. The amino acid sequence and secondary structure of the Nectria TAm. The residues proposed by consensus analysis to be important for (R)-selective amine activity are marked 22. Motif 1 (blue stars): H(53); Y(58); V(60) and S(62). Motif 2 (red stars): F(113); V(114); E(115); V(125); R(126); G(127); A(128). The active site Lys179 is highlighted by a green star. The secondary structural elements are indicated above the sequence, respectively, as α-helices, η-310 helices and β-strands. The N-terminal domain β-sheet is shown as βA–βH, the PLP binding domain β-sheet as β1–β9. The β-strand forming the inter-subunit β-strand with its symmetry equivalent is marked as β*. The secondary structure assignment and the figure were produced using espript
45.
Figure 7. SchemeSpectophotometric assay used to screen the Nectria TAm with several chiral amines.
Figure 8. SchemeAsymmetric reductive amination of 1a, 3a, 5a catalysed by Nectria TAm with simultaneous co-product removal using lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH) and NAD+ as cofactor.
Figure 2. (A) A cartoon diagram of the Nectria TAm dimer viewed normal to its molecular dyad; the individual subunits are shown in blue and purple. The mCPP adduct is shown as a space-filling model. (B) A cartoon diagram of the Nectria TAm subunit with the PLP binding domain and the N-terminal domain coloured purple and red respectively. The interdomain loop region 146–151 is shown in black. The secondary structure elements are numbered, the N-terminal domain strands are labelled βA–βH, and the PLP binding domain strands β1–β8. The mCPP adduct is shown as sticks indicating the location of the active site cavity.
Figure 3. (A) A stereo diagram showing the Nectria TAm active site with the bound mCPP adduct. Residues forming the L and S binding pockets are shown as sticks. (B) The 2Fo − Fc electron density map (blue) was contoured at 1 σ for the gabaculine-PLP bound adduct and neighbouring residues. The positive Fo − Fc electron density map is shown in green and negative in red with both contoured at 3.1 σ. The two conformations of the Phe113 induced by the mCPP binding are shown with the disorder of the carboxyl group of mCPP highlighted by the negative Fo − Fc density.
Figure 4. A stereo diagram showing the structural basis of the (R)-enantioselectivity of the Nectria TAm. Both enantiomers of MBA were positioned for catalysis based on the structure of the mCPP complex and the requirement for the scissile Cα-H bond to be normal to the plane of the pyridine ring of PLP. The (S)-MBA enantiomer in this position is pointing into the S pocket where it is sterically hindered by residues Thr273-Ala275.
Figure 5. A stereo diagram showing the superposition of the dimeric Nectria TAm structure (subunits are shown as green and red cartoons) with the Bacillus sp. DATA structure (magenta and cyan cartoons). This is viewed from the solvent region into the active site cavity. The Arg98 binds the α-carboxyl group of the substrate in DATA as shown for the complex with the reaction intermediate analogue pyridoxyl-d-alanine (PDD; PDB code 3DAA). In BCAT this arginine is not conserved, although the equivalent loop retains similar conformation (not shown). In the Nectria TAm the corresponding loop 122–136, which contains four residues of the second fingerprint sequence motif, accepts a distinct conformation with part of it folding into 310 helix. This results in significant displacement of conserved Arg126, which is unable to bind the substrate analogue mCPP carboxyl group since it is too remote. The ligands and the residues are shown as stick models.
Altschul,
Basic local alignment search tool.
1990, Pubmed
Altschul,
Basic local alignment search tool.
1990,
Pubmed
Cho,
Asymmetric synthesis of L-homophenylalanine by equilibrium-shift using recombinant aromatic L-amino acid transaminase.
2003,
Pubmed
Coleman,
The genome of Nectria haematococca: contribution of supernumerary chromosomes to gene expansion.
2009,
Pubmed
,
Echinobase
Dunathan,
Conformation and reaction specificity in pyridoxal phosphate enzymes.
1966,
Pubmed
Emsley,
Features and development of Coot.
2010,
Pubmed
Fuchs,
Chemoenzymatic asymmetric total synthesis of (S)-Rivastigmine using omega-transaminases.
2010,
Pubmed
Gefflaut,
Preparation of glutamate analogues by enzymatic transamination.
2012,
Pubmed
Gouet,
ESPript: analysis of multiple sequence alignments in PostScript.
1999,
Pubmed
Hayashi,
Pyridoxal enzymes: mechanistic diversity and uniformity.
1995,
Pubmed
Hopwood,
A fast and sensitive assay for measuring the activity and enantioselectivity of transaminases.
2011,
Pubmed
Humble,
Crystal structures of the Chromobacterium violaceumω-transaminase reveal major structural rearrangements upon binding of coenzyme PLP.
2012,
Pubmed
Höhne,
Rational assignment of key motifs for function guides in silico enzyme identification.
2010,
Pubmed
Jansonius,
Structure, evolution and action of vitamin B6-dependent enzymes.
1998,
Pubmed
John,
Pyridoxal phosphate-dependent enzymes.
1995,
Pubmed
Kabsch,
XDS.
2010,
Pubmed
Langer,
Visual automated macromolecular model building.
2013,
Pubmed
Leuchtenberger,
Biotechnological production of amino acids and derivatives: current status and prospects.
2005,
Pubmed
Malik,
Features and technical applications of ω-transaminases.
2012,
Pubmed
Mehta,
Aminotransferases: demonstration of homology and division into evolutionary subgroups.
1993,
Pubmed
Murshudov,
REFMAC5 for the refinement of macromolecular crystal structures.
2011,
Pubmed
Okada,
Structures of Escherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine: induced fit and substrate recognition of the enzyme.
2001,
Pubmed
Peisach,
Crystallographic study of steps along the reaction pathway of D-amino acid aminotransferase.
1998,
Pubmed
Punta,
The Pfam protein families database.
2012,
Pubmed
Rando,
Mechanism of the irreversible inhibition of gamma-aminobutyric acid-alpha-ketoglutaric acid transaminase by the neutrotoxin gabaculine.
1977,
Pubmed
Savile,
Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture.
2010,
Pubmed
Sayer,
Crystal structure and substrate specificity of the thermophilic serine:pyruvate aminotransferase from Sulfolobus solfataricus.
2012,
Pubmed
Sayer,
Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity.
2013,
Pubmed
Shin,
Purification, characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17.
2003,
Pubmed
Stewart,
Dehydrogenases and transaminases in asymmetric synthesis.
2001,
Pubmed
Studier,
Protein production by auto-induction in high density shaking cultures.
2005,
Pubmed
Taylor,
Novel biosynthetic approaches to the production of unnatural amino acids using transaminases.
1998,
Pubmed
Thomsen,
Crystallization and preliminary X-ray diffraction studies of the (R)-selective amine transaminase from Aspergillus fumigatus.
2013,
Pubmed
Tufvesson,
Process considerations for the asymmetric synthesis of chiral amines using transaminases.
2011,
Pubmed
Vagin,
Molecular replacement with MOLREP.
2010,
Pubmed
Vaguine,
SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model.
1999,
Pubmed
Winn,
Overview of the CCP4 suite and current developments.
2011,
Pubmed
Winter,
Decision making in xia2.
2013,
Pubmed