ARTICLES
containing 0.1 mM PLP. The purified enzyme solutions were stored at +4 °C, or at
−20 °C in 30% glycerol.
11. Han, S.-W., Park, E.-S., Dong, J.-Y. & Shin, J.-S. Mechanism-guided engineering
of ω-transaminase to accelerate reductive amination of ketones. Adv. Synth.
Catal. 357, 1732–1740 (2015).
Determination of activity in kinetic resolution mode. The activity measurements
were performed in a TECAN Infinite 200 PRO reader, and all measurements were
performed at least in triplicate. A direct photometric assay was used28, in which the
enzymatic reaction took place in CHES buffer (50 mM, pH 9.0) at 30 °C in a total
volume of 200 µl. 1 mM racemic amine (1a–4a) and 2 mM pyruvate were added to the
buffer (final concentrations) containing the TA of interest. Due to the preparation of
the amine stock solution in organic solvent, 5% (vol/vol) 2-propanol or
12. Sayer, C. et al. The substrate specificity, enantioselectivity and structure of the
(R)-selective amine: pyruvate transaminase from Nectria haematococca. FEBS J.
281, 2240–2253 (2014).
13. Jiang, J., Chen, X., Feng, J., Wu, Q. & Zhu, D. Substrate profile of an
ω-transaminase from Burkholderia vietnamiensis and its potential for the
production of optically pure amines and unnatural amino acids. J. Mol. Catal. B
100, 32–39 (2014).
dimethylsulfoxide (DMSO, 0.1% for compounds 5–7) was present in the final reaction
medium. The kinetic resolution was initiated by the addition of the amine acceptor
14. Nobili, A. et al. Engineering the active site of the amine transaminase from
Vibrio fluvialis for the asymmetric synthesis of aryl–alkyl amines and amino
alcohols. ChemCatChem 7, 757–760 (2015).
(pyruvate). The final concentration of the enzyme varied between 2 and 800 µg ml−1
,
depending on the specific activity towards the substrates under examination. The
production of the corresponding ketones (1b–7b) was monitored at the optimum
wavelength for each compound (1b, 16,562 M−1 cm−1 at 264 nm; 2b, 9,646 M−1 cm−1
at 245 nm; 3b, 5,714 M−1 cm−1 at 340 nm; 4b, 6,115 M−1 cm−1 at 245 nm (ref. 27);
5b and 6b, 6,530 M−1 cm−1 at 242 nm (ref. 15); and 7b, 7,098 M−1 cm−1
at 252 nm (ref. 14)).
15. Genz, M. et al. Alteration of the donor/acceptor spectrum of the (S)-amine
transaminase from Vibrio fluvialis. Int. J. Mol. Sci. 16, 26953–26963 (2015).
16. Steffen-Munsberg, F. et al. Connecting unexplored protein crystal structures to
enzymatic function. ChemCatChem 5, 150–153 (2013).
17. Middelfort, K. S. et al. Redesigning and characterizing the substrate specificity
and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin.
Protein Eng. Des. Sel. 26, 25–33 (2013).
Preparative asymmetric synthesis. The starting volume of all reactions was 100 ml
and the substrate amount 100 mg ketone. The reaction mixture also contained
HEPES buffer (50–70 mM) including 1 mM PLP, DMSO (20% (vol/vol) for 1b–3b
or 2% (vol/vol) for 5b–7b), D,L-alanine (200–275 mM), oxidized nicotinamide
adenine dinucleotide (NAD+, 2–3 mM), GDH (3 U ml−1), D-glucose (220 mM) and
LDH (6 U ml−1). The slight variations in the reagent concentrations depended on
the applied amount of purified enzyme solution (protein applied: 57 mg for
synthesis of (R)-1a, 20 mg for (S)-2a and 39 mg for (S)-3a) or 100 mg enzyme
lyophilisate (66 mg protein for the synthesis of (S)-5a, (S)-6a and (R)-7a). The
addition of the respective ketone as DMSO solution started the reaction at pH 8.0
and 30 °C. The pH of the stirred reaction was kept constant by the addition of 0.1 N
NaOH until full conversion was indicated by HPLC (Table 3). Afterwards, the
reaction was quenched with HCl to pH 2.0 and the precipitated proteins could be
removed by dicalite treatment and filtration. The products were extracted with
tert-butyl methyl ether (TBME) at pH 10 (NaOH addition), delivering (after drying
and evaporation) the crude amines as oils. These were characterized (NMR, MS,
chiral and achiral HPLC) either as their HCl salts (1–3a) or as free amines (5–7a).
For amine 3a, salt formation was essential due to the instability of the free base,
which degraded, with discolouration, fairly rapidly, most probably due to oxidative
aromatization. Secondary product formation was suppressed further by applying a
nitrogen atmosphere, minimizing the risk of oxidation by air. For amine 7a, the
aqueous solution needed to be saturated with NaCl before its extraction with TBME.
18. Kaulmann, U., Smithies, K., Smith, M. E. B., Hailes, H. C. & Ward, J. M. Substrate
spectrum of ω-transaminase from Chromobacterium violaceum DSM30191 and
its potential for biocatalysis. Enzyme Microb. Technol. 41, 628–637 (2007).
19. Cassimjee, K. E., Humble, M. S., Land, H., Abedi, V. & Berglund, P.
Chromobacterium violaceum ω-transaminase variant Trp60Cys shows increased
specificity for (S)-1-phenylethylamine and 4′-substituted acetophenones, and
follows Swain–Lupton parameterisation. Org. Biomol. Chem. 10, 5466–5470 (2012).
20. Steffen-Munsberg, F. et al. Revealing the structural basis of promiscuous amine
transaminase activity. ChemCatChem 5, 154–157 (2013).
21. Cassimjee, K. E., Manta, B. & Himo, F. A quantum chemical study of the
ω-transaminase reaction mechanism. Org. Biomol. Chem. 13, 8453–8464 (2015).
22. Yu, H., Zhao, Y., Guo, C., Gan, Y. & Huang, H. The role of proline substitutions
within flexible regions on thermostability of luciferase. Biochim. Biophys. Acta
Prot. Proteomics 1854, 65–72 (2015).
23. Deszcz, D. et al. Single active-site mutants are sufficient to enhance serine:pyruvate
α-transaminase activity in an ω-transaminase. FEBS J. 282, 2512–2526 (2015).
24. Wilke, A. et al. The M5nr: a novel non-redundant database containing protein
sequences and annotations from multiple sources and associated tools.
BMC Bioinformatics 13, 141 (2012).
25. Levin, K. B. et al. Following evolutionary paths to protein–protein interactions
with high affinity and selectivity. Nature Struct. Mol. Biol. 16, 1049–1055 (2009).
26. Shafee, T., Gatti-Lafranconi, P., Minter, R. & Hollfelder, F. Handicap-recover
evolution leads to a chemically versatile, nucleophile-permissive protease.
ChemBioChem 16, 1866–1869 (2015).
Received 4 February 2016; accepted 16 June 2016;
published online 18 July 2016
27. Grishin, N. V., Phillips, M. A. & Goldsmith, E. J. Modeling of the spatial structure
of eukaryotic ornithine decarboxylases. Protein Sci. 4, 1291–1304 (1995).
28. Schätzle, S., Höhne, M., Redestad, E., Robins, K. & Bornscheuer, U. T. Rapid and
sensitive kinetic assay for characterization of ω-transaminases. Anal. Chem. 81,
8244–8248 (2009).
References
1. Truppo, M. D., Rozzell, J. D. & Turner, N. J. Efficient production of
enantiomerically pure chiral amines at concentrations of 50 g/L using
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ketones applied to sitagliptin manufacture. Science 329, 305–309 (2010).
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superfamily suitable for biocatalytic applications. Biotechnol. Adv. 33, 566–604 (2015).
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Acknowledgements
The authors thank J.F. Kabisch for preparing the M5nr database, M. Althaus and I. Duffour
for developing the chiral and achiral analysis methods, J. Joerger and M. Rothe for the
preparative separation of the chiral amines, C. Wyss-Gramberg for the NMR analysis of the
Mosher amides, I. Menyes for support with HPLC and gas chromatography analyses and
P. Meier for performing the preparative asymmetric synthesis experiments.
Author contributions
U.T.B., H.I. and B.W. initiated the study and directed the project. P.S. undertook the
substrate and product syntheses. I.V.P. performed the bioinformatics analysis. I.V.P.,
M.S.W. and M.G. prepared and characterized all the variants. I.V.P, S.P.H. and H.I.
performed the preparative asymmetric synthesis experiments. I.V.P., H.I. and U.T.B.
prepared the manuscript, which was revised and approved by all authors.
Additional information
requests for materials should be addressed to H.I. and U.T.B.
Competing financial interests
10. Desai, A. A. Sitagliptin manufacture: a compelling tale of green chemistry,
process intensification, and industrial asymmetric catalysis. Angew. Chem. Int.
Ed. 50, 1974–1976 (2011).
The biocatalysis group of Roche has a committed interest over the long term in establishing a set
of technically applicable TAs with broad substrate acceptance to assist devising more attractive,
shorter, economical and greener synthetic routes to investigational drugs and beyond.
7
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