TY - JOUR
T1 - Mutagenesis-Independent Stabilization of Class B Flavin Monooxygenases in Operation
AU - Goncalves, Leticia C.P.
AU - Kracher, Daniel
AU - Milker, Sofia
AU - Fink, Michael J.
AU - Rudroff, Florian
AU - Ludwig, Roland
AU - Bommarius, Andreas S.
AU - Mihovilovic, Marko D.
N1 - Funding Information:
We thank David Siebert (TU Wien) for the purification of FAD, Dr. Anna Münch (NanoTemper Technologies GmbH) for generously providing access to the DSF fluorimeter, and Prof. Marco W. Fraaije (University of Groningen) for kindly providing a sample of the CHMO-PTDH fusion enzyme. This work was supported by the Austrian Science Fund FWF (grants no. I723-N17 and P24483-B20), by the COST action Systems Biocatalysis WG2, by TU Wien (ABC-Top Anschubfinanzierung), by the European Commission Project INDOX (grant no. FP7-KBBE-2013-7-613549), and by the Coordination for the Improvement of Higher Education Personnel (grant no. CAPES/CsF, 2505-13-4).
Publisher Copyright:
© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
PY - 2017/6/19
Y1 - 2017/6/19
N2 - This paper describes the stabilization of flavin-dependent monooxygenases under reaction conditions, using an engineered formulation of additives (the natural cofactors NADPH and FAD, and superoxide dismutase and catalase as catalytic antioxidants). This way, a 103- to 104-fold increase of the half-life was reached without resource-intensive directed evolution or structure-dependent protein engineering methods. The stabilized enzymes are highly valued for their synthetic potential in biotechnology and medicinal chemistry (enantioselective sulfur, nitrogen and Baeyer–Villiger oxidations; oxidative human metabolism), but widespread application was so far hindered by their notorious fragility. Our technology immediately enables their use, does not require structural knowledge of the biocatalyst, and creates a strong basis for the targeted development of improved variants by mutagenesis. (Figure presented.).
AB - This paper describes the stabilization of flavin-dependent monooxygenases under reaction conditions, using an engineered formulation of additives (the natural cofactors NADPH and FAD, and superoxide dismutase and catalase as catalytic antioxidants). This way, a 103- to 104-fold increase of the half-life was reached without resource-intensive directed evolution or structure-dependent protein engineering methods. The stabilized enzymes are highly valued for their synthetic potential in biotechnology and medicinal chemistry (enantioselective sulfur, nitrogen and Baeyer–Villiger oxidations; oxidative human metabolism), but widespread application was so far hindered by their notorious fragility. Our technology immediately enables their use, does not require structural knowledge of the biocatalyst, and creates a strong basis for the targeted development of improved variants by mutagenesis. (Figure presented.).
KW - biocatalysis
KW - cofactors
KW - enzyme stabilization
KW - oxygenation
KW - reactive oxygen species
UR - http://www.scopus.com/inward/record.url?scp=85020904419&partnerID=8YFLogxK
U2 - 10.1002/adsc.201700585
DO - 10.1002/adsc.201700585
M3 - Article
AN - SCOPUS:85020904419
SN - 1615-4150
VL - 359
SP - 2121
EP - 2131
JO - Advanced Synthesis and Catalysis
JF - Advanced Synthesis and Catalysis
IS - 12
ER -